Engineered Artificial Antigen Presenting Cells for Tumor Infiltrating Lymphocyte Expansion

ABSTRACT

In some embodiments, compositions and methods relating to isolated artificial antigen presenting cells (aAPCs) are disclosed, including aAPCs comprising a myeloid cell transduced with one or more viral vectors, such as a MOLM-14 or a EM-3 myeloid cell, wherein the myeloid cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-1BBL and/or OX40L proteins. In some embodiments, methods of expanding tumor infiltrating lymphocytes (TILs) with aAPCs and methods of treating cancers using TILs after expansion with aAPCs are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/526,353, filed on Jul. 30, 2019, which is a continuation of U.S.patent application Ser. No. 15/800,967, filed Nov. 1, 2017, which is acontinuation of International Application No. PCT/US17/59271, filed Oct.31, 2017, which claims the benefit of priority to U.S. ProvisionalApplication No. 62/481,831, filed Apr. 5, 2017, U.S. ProvisionalApplication No. 62/475,053, filed Mar. 22, 2017, U.S. ProvisionalApplication No. 62/438,600, filed Dec. 23, 2016, and U.S. ProvisionalApplication No. 62/415,274, filed Oct. 31, 2016, the entireties of whichare incorporated herein by reference.

FIELD OF THE INVENTION

Engineered artificial antigen presenting cells (aAPCs) for expansion oftumor infiltrating lymphocytes are disclosed.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive autologoustransfer of tumor infiltrating lymphocytes (TILs) represents a powerfulapproach to therapy for patients with poor prognoses. Gattinoni, et al.,Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs are requiredfor successful immunotherapy, and a robust and reliable process isneeded for commercialization. This has been a challenge to achievebecause of technical, logistical, and regulatory issues with cellexpansion. IL-2-based TIL expansion followed by a “rapid expansionprocess” (REP) has become a preferred method for TIL expansion becauseof its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54;Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257,238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. However,although REP can result in a 1,000-fold expansion of TILs over a 14-dayperiod, it requires a large excess (e.g., 200-fold) of irradiatedallogeneic peripheral blood mononuclear cells (PBMCs), often frommultiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3)and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42.Despite their high performance, PBMCs have multiple drawbacks, includingthe large numbers of allogeneic PBMCs required, the need to obtain PBMCsby leukapheresis from multiple healthy donors, the resulting interdonorvariability in PBMC viability after cryopreservation and variable TILexpansion results, the risk of undetected viral pathogens causingdownstream patient infections, and the extensive and costly laboratorytesting of each individual donor cell product to confirm sterility andquality (including viral contaminant testing) and to test expansionproperties.

Unfortunately, aAPCs developed for use in the expansion of TILs havesuffered from poor performance when compared to PBMCs, includingalterations of the phenotypic properties of the input TILs, as well aspoor expansion performance and/or high variability in expansion results.Because of the large number of potential cells that might be adapted foruse as aAPCs and the unpredictability of identifying suitablecandidates, the focus of aAPC development for polyclonal TILs to datehas been solely on the well-established K562 cell line. Butler andHirano, Immunol. Rev. 2014, 257, 191-209. For example, K562 cellsmodified to express 4-1BBL (CD137L) were tested in pre-REP culture (butnot in REP culture) to determine enhancement of TIL expansion from tumordigest, but PBMCs were still required to be used in conjunction withK562 cells to obtain TIL expansion. Friedman, et al., J. Immunother.2011, 34, 651-661. Other engineered K562 cells modified to express CD64,CD86, and 4-1BBL were tested and achieved TIL expansion that was at bestcomparable to PBMCs, and most likely less than PBMCs, and also sufferedfrom skewing of the polyclonal TIL phenotype to a less favorableCD8⁺/CD4⁺ T cell ratio. Ye, et al., J. Translat. Med. 2011, 9, 131.Recently, K562 cells modified to express CD86, 4-1BBL (CD137L), highaffinity Fc receptor (CD64) and membrane-bound IL-15 have also beenshown to propagate TIL (post-REP) at equivalent numbers compared to PBMCfeeders, but with the additional complexity of membrane-bound IL-15.Forget, et al., J. Immunother. 2014, 37, 448-60. Other systems developedhave lacked critical costimulatory molecules, have led to unfavorable Tcell phenotypic skewing, or have required additional interleukins (suchas IL-21). Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. Overall,K562 modified aAPCs have not been shown to provide for consistentexpansion of TILs with acceptable variability while also performingbetter than PBMCs in other measures including overall expansion cellcounts. Alternative aAPCs besides K562 cells have been successful inother cell expansion methods, but have not achieved the same performanceas PBMCs with the unique polyclonal subset of cells that make up TILs.Maus, et al., Nat. Biotechnol. 2002, 20, 143-148; Suhoski, et al., MotTher. 2007, 15, 981-988.

The MOLM-14 human leukemia cell line was established from the peripheralblood of a patient with relapsed acute monocytic leukemia, and initialphenotypic characterization indicated the presence of at least thefollowing markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64,CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al.,Leukemia 1997, 11, 1469-77. Additional phenotypic characterization ofMOLM-14 found higher levels of HLA-A/B/C, CD64, CD80, ICOS-L, CD58, andlower levels of CD86. MOLM-14 cells and the closely-related MOLM-13cells have not been previously reported as useful aAPCs for theexpansion of cells for tumor immunotherapy applications.

The EM-3 human cell line was established from the bone marrow of apatient with Philadelphia chromosome-positive CIVIL. Konopka, et al.,Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. EM-3 cells and theclosely-related EM-2 cell line have not been previously reported asuseful aAPCs for the expansion of cells for tumor immunotherapyapplications. Phenotypic characterization for EM-3 cells indicates thepresence of at least the following markers: CD13, CD15, and CD33.

The present invention provides the unexpected finding that engineeredmyeloid lineage cells, including MOLM-13, MOLM-14, EM-3, and EM-2 cells,transduced with additional costimulatory molecules, including CD86(B7-2), 4-1BBL (CD137L), and OX40L (CD134L), provide for superior andhighly efficient expansions of TILs in large numbers with minimalvariability, reduced cost, and no reliance on human blood samples as asource of PBMCs, with the benefit of using an aAPC which can be producedefficiently from a master cell bank. CD86 and 4-1BBL are costimulatorymolecules that provide costimulatory signals for T cell activation. TheMOLM-14, MOLM-13, EM-3, and/or EM-2 cells transduced with additionalcostimulatory molecules are useful, for example, in the expansion ofTILs for use in cancer immunotherapy and other therapies.

SUMMARY OF THE INVENTION

In an embodiment, the invention provides an artificial antigenpresenting cell (aAPC) comprising a myeloid cell transduced with one ormore vectors, wherein the one or more viral vectors comprise a nucleicacid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL,and wherein the myeloid cell expresses a CD86 protein and a 4-1BBLprotein.

In an embodiment, each of the CD86 protein and the 4-1BBL protein arehuman proteins.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the aAPC canstimulate and expand a tumor infiltrating lymphocyte (TIL) contactedwith the aAPC.

It will be apparent that in certain embodiments of the invention, thenucleic acid molecule encoding CD86 may be comprised in a differentviral vector to the nucleic acid molecule encoding 4-1BBL or the sameviral vector.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the aAPC expandsa population of TILs by at least 50-fold over a period of 7 days in acell culture medium comprising IL-2 at a concentration of about 3000IU/mL and OKT-3 antibody at a concentration of about 30 ng/mL.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the aAPC canstimulate and expand a T cell contacted with the aAPC.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellendogenously expresses HLA-AB/C, ICOS-L, and CD58.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellis essentially devoid of membrane-bound IL-15.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellis a MOLM-14 cell.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellis a MOLM-13 cell.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellis a EM-3 cell.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cellis a EM-2 cell.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the CD86 proteincomprises an amino acid sequence as set forth in SEQ ID NO:8, or anamino acid sequence comprising one or more conservative amino acidsubstitutions thereof, and the 4-1BBL protein comprises SEQ ID NO:9, oran amino acid sequence comprising one or more conservative amino acidsubstitutions thereof.

In an embodiment, the invention provides an aAPC comprising a myeloidcell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid molecule encoding CD86 and anucleic acid molecule encoding 4-1BBL, and wherein the myeloid cellexpresses a CD86 protein and a 4-1BBL protein, wherein the nucleic acidmolecule encoding CD86 comprises a nucleic acid sequence as set forth inSEQ ID NO:16 and the nucleic acid molecule encoding 4-1BBL comprises anucleic acid sequence as set forth in SEQ ID NO:19.

In an embodiment, the invention provides a method of expanding tumorinfiltrating lymphocytes (TILs), the method comprising the step ofcontacting a population of TILs with an aAPC comprising a myeloid celltransduced with one or more viral vectors, wherein the one or more viralvectors comprise a nucleic acid molecule encoding CD86 and a nucleicacid molecule encoding 4-1BBL, wherein the myeloid cell expresses a CD86protein and a 4-1BBL protein, and wherein the population of TILs isexpanded. In an embodiment, the method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the cell culture medium        further comprises IL-2 at an initial concentration of about 3000        IU/mL and OKT-3 antibody at an initial concentration of about 30        ng/mL.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the population of APCs        expands the population of TILs by at least 50-fold over a period        of 7 days in a cell culture medium.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the myeloid cell        endogenously expresses HLA-AB/C, ICOS-L, and CD58.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the myeloid cell is a        MOLM-14 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the myeloid cell is a        MOLM-13 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the myeloid cell is a        EM-3 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the myeloid cell is a        EM-2 cell.

In an embodiment, the foregoing method is an in vitro or an ex vivomethod.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid molecule encoding CD86 and a nucleic acid molecule        encoding 4-1BBL, and wherein the myeloid cell expresses a CD86        protein and a 4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the CD86 protein        comprises an amino acid sequence as set forth in SEQ ID NO:8, or        comprises an amino acid sequence comprising one or more        conservative amino acid substitutions thereof, and the 4-1BBL        protein comprises an amino acid sequence as set forth in SEQ ID        NO:9, or comprises an amino acid sequence comprising one or        conservative amino acid substitutions thereof.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the nucleic acid        encoding CD86 comprises a nucleic acid sequence as set forth in        SEQ ID NO:16 and the nucleic acid encoding 4-1BBL comprises a        nucleic acid sequence as set forth in SEQ ID NO:19.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the expansion is        performed using a gas permeable container.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the ratio of the        population of TILs to the population of aAPCs is between 1 to        200 and 1 to 400.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium, wherein the ratio of the        population of TILs to the population of aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of expanding tumorinfiltrating lymphocytes (TILs), the method comprising contacting apopulation of TILs comprising a population of TILs with a myeloidartificial antigen presenting cell (aAPC), wherein the myeloid aAPCcomprises at least two co-stimulatory ligands that specifically bindwith at least two co-stimulatory molecules on the TILs, wherein bindingof the co-stimulatory molecules with the co-stimulatory ligand inducesproliferation of the TILs, thereby specifically expanding TILs, andwherein the at least two co-stimulatory ligands comprise CD86 and4-1BBL. In an embodiment, the foregoing method is an in vitro or ex vivomethod.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced        to express a CD86 protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating cancer, wherein theTILs are a second population of TILs and are obtainable from a methodcomprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the TILs are/have been        obtained from a tumor resected from a patient, and wherein the        second population of TILs is at least 50-fold greater in number        than the first population of TILs after 7 days from the start of        the rapid expansion; and    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced        to express a CD86 protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the myeloid        aAPCs comprise MOLM-14 cells transduced with one or more viral        vectors, and wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the MOLM-14 cells express a CD86 protein and a        4-1BBL protein.

In an embodiment, the invention provides a population of tumorinfiltrating cells (TILs) for use in treating a cancer, wherein thepopulation of TILs is a second population of TILs and is obtainable by aprocess comprising:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        are/have been obtained from a tumor resected from a patient,        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the myeloid        aAPCs comprise MOLM-14 cells transduced with one or more viral        vectors, and wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the MOLM-14 cells express a CD86 protein and a        4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the myeloid        aAPCs comprise EM-3 cells transduced with one or more viral        vectors, and wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the EM-3 cells express a CD86 protein and a 4-1BBL        protein.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, thepopulation of TILs being a second population of TILs and obtainable by aprocess comprising:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        are/have been obtained from a tumor resected from a patient, and        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion; and    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the myeloid        aAPCs comprise EM-3 cells transduced with one or more viral        vectors, and wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the EM-3 cells express a CD86 protein and a 4-1BBL        protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        rapid expansion is performed over a period not greater than 14        days.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a second population and is obtainable by amethod comprising the steps of:

-   -   (a) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion,        wherein the myeloid aAPCs endogenously express HLA-AB/C, ICOS-L        and CD58, wherein the myeloid aAPCs are transduced to express a        CD86 protein and a 4-1BBL protein, and wherein the rapid        expansion is performed over a period not greater than 14 days.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        cell culture medium further comprises IL-2 at an initial        concentration of about 3000 IU/mL and OKT-3 antibody at an        initial concentration of about 30 ng/mL.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, thepopulation of TILs being a second population of TILs and obtainable by aprocess comprising:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        are/have been obtained from a tumor resected from a patient, and        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion; and wherein the myeloid        aAPCs endogenously express HLA-AB/C, ICOS-L, and CD58, wherein        the myeloid aAPCs are transduced to express a CD86 protein and a        4-1BBL protein, and wherein the cell culture medium further        comprises IL-2 at an initial concentration of about 3000 IU/mL        and OKT-3 antibody at an initial concentration of about 30        ng/mL.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        expansion is performed using a gas permeable container.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, thepopulation of TILs being a second population of TILs and obtainable by aprocess comprising:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        are/have been obtained from a tumor resected from a patient, and        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion; and wherein the myeloid        aAPCs endogenously express HLA-AB/C, ICOS-L, and CD58, wherein        the myeloid aAPCs are transduced to express a CD86 protein and a        4-1BBL protein, and wherein the expansion is performed using a        gas permeable container.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        ratio of the second population of TILs to the population of        aAPCs is between 1 to 200 and 1 to 400.

In an embodiment, the invention provides a population of tumorinfiltrating cells (TILs) for use in treating a cancer, the populationof TILs being a second population of TILs and obtainable by a processcomprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        is/has been obtained from a tumor resected from a patient, and        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion; and    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        ratio of the second population of TILs to the population of        aAPCs is between 1 to 200 and 1 to 400. In certain embodiments,        the ratio of the second population of TILs to the population of        aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, and wherein the        ratio of the second population of TILs to the population of        aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing a rapid expansion of the first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain a        second population of TILs, wherein the second population of TILs        is at least 50-fold greater in number than the first population        of TILs after 7 days from the start of the rapid expansion; and    -   (c) administering a therapeutically effective portion of the        second population of TILs to a patient with the cancer;    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the cancer        is selected from the group consisting of melanoma, ovarian        cancer, cervical cancer, non-small-cell lung cancer (NSCLC),        lung cancer, bladder cancer, breast cancer, cancer caused by        human papilloma virus, head and neck cancer, renal cancer, and        renal cell carcinoma.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, thepopulation of TILs being a second population of TILs and obtainable by amethod comprising the steps of:

-   -   (a) performing a rapid expansion of a first population of TILs        using a population of myeloid artificial antigen presenting        cells (myeloid aAPCs) in a cell culture medium to obtain the        second population of TILs, wherein the first population of TILs        is/has been obtained from a tumor resected from a patient, and        wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs after 7 days        from the start of the rapid expansion; and    -   wherein the myeloid aAPCs endogenously expresses HLA-AB/C,        ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to        express a CD86 protein and a 4-1BBL protein, wherein the cancer        is selected from the group consisting of melanoma, ovarian        cancer, cervical cancer, non-small-cell lung cancer (NSCLC),        lung cancer, bladder cancer, breast cancer, cancer caused by        human papilloma virus, head and neck cancer, renal cancer, and        renal cell carcinoma.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the myeloid aAPCs comprise MOLM-14 cells transduced with        one or more viral vectors, wherein the one or more viral vectors        comprise a nucleic acid encoding CD86 and a nucleic acid        encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86        protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the myeloid aAPCs comprise EM-3 cells transduced with        one or more viral vectors, wherein the one or more viral vectors        comprise a nucleic acid encoding CD86 and a nucleic acid        encoding 4-1BBL, and wherein the EM-3 cells express a CD86        protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a third population of TILs and obtainable by amethod comprising the steps of:

-   -   (a) performing an initial expansion of a first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first population of TILs is/has        been obtained from a tumor resected from a patient, and wherein        the second population of TILs is at least 5-fold greater in        number than the first population of TILs, and wherein the first        cell culture medium comprises IL-2;    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3.

In an embodiment, the myeloid aAPCs comprise MOLM-14 cells transducedwith one or more viral vectors, wherein the one or more viral vectorscomprise a nucleic acid encoding CD86 and a nucleic acid encoding4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a4-1BBL protein. In an embodiment, the myeloid cells comprise MOLM-13cells transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 proteinand a 4-1BBL protein. In certain embodiments, the myeloid cells compriseEM-3 cells transduced with one or more viral vectors, wherein the one ormore viral vectors comprise a nucleic acid encoding CD86 and a nucleicacid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 proteinand a 4-1BBL protein. In certain embodiments, the myeloid cells compriseEM-2 cells transduced with one or more viral vectors, wherein the one ormore viral vectors comprise a nucleic acid encoding CD86 and a nucleicacid encoding 4-1BBL, and wherein the EM-2 cells express a CD86 proteinand a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) treating the patient with a non-myeloablative        lymphodepletion regimen, wherein the non-myeloablative        lymphodepletion regimen comprises the steps of administration of        cyclophosphamide at a dose of 60 mg/m²/day for two days followed        by administration of fludarabine at a dose of 25 mg/m²/day for        five days;    -   (e) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer; and    -   (f) treating the patient with a high-dose IL-2 regimen, wherein        the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of        aldesleukin administered as a 15-minute bolus intravenous        infusion every eight hours until tolerance;    -   wherein the myeloid aAPCs comprise MOLM-14 cells transduced with        one or more viral vectors, wherein the one or more viral vectors        comprise a nucleic acid encoding CD86 and a nucleic acid        encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86        protein and a 4-1BBL protein.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) treating the patient with a non-myeloablative        lymphodepletion regimen, wherein the non-myeloablative        lymphodepletion regimen comprises the steps of administration of        cyclophosphamide at a dose of 60 mg/m²/day for two days followed        by administration of fludarabine at a dose of 25 mg/m²/day for        five days;    -   (e) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer; and    -   (f) treating the patient with a high-dose IL-2 regimen, wherein        the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of        aldesleukin administered as a 15-minute bolus intravenous        infusion every eight hours until tolerance;    -   wherein the myeloid aAPCs comprise EM-3 cells transduced with        one or more viral vectors, wherein the one or more viral vectors        comprise a nucleic acid encoding CD86 and a nucleic acid        encoding 4-1BBL, and wherein the EM-3 cells express a CD86        protein and a 4-1BBL protein.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs are a third population of TILs and obtainable bya method comprising the steps of:

-   -   (a) an initial expansion of a first population of TILs in a        first cell culture medium to obtain a second population of TILs,        wherein the first population of TILs is/has been obtained from a        tumor resected from a patient, and wherein the second population        of TILs is at least 5-fold greater in number than the first        population of TILs, and wherein the first cell culture medium        comprises IL-2; and    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        and further wherein the population of TILs is for administration        to a patient in combination with a non-myeloablative        lymphodepletion regimen, wherein the non-myeloablative        lymphodepletion regimen comprises cyclophosphamide which is for        administration at a dose of 60 mg/m²/day for two days followed        by fludarabine which is for administration at a dose of 25        mg/m²/day for five days and further wherein the population of        TILs is for administration in combination with a high-dose IL-2        regimen, wherein the high-dose IL-2 regimen comprises 600,000 or        720,000 IU/kg of aldesleukin for administration as a 15-minute        bolus intravenous infusion every eight hours until tolerance. In        certain embodiments, the population of TILs is for        administration prior to the high-dose IL-2 regimen and        subsequent to the non-myeloablative lymphodepletion regimen.

In certain embodiments, the myeloid aAPCs comprise MOLM-14 cellstransduced with one or more viral vectors, wherein the one or more viralvectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 proteinand a 4-1BBL protein. the myeloid aAPCs comprise MOLM-13 cellstransduced with one or more viral vectors, wherein the one or more viralvectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 proteinand a 4-1BBL protein. In certain embodiments, the myeloid aAPCs compriseEM-3 cells transduced with one or more viral vectors, wherein the one ormore viral vectors comprise a nucleic acid encoding CD86 and a nucleicacid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 proteinand a 4-1BBL protein.

In an embodiment, the population of TILs is for use in the treating of acancer selected from the group consisting of melanoma, ovarian cancer,cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer,bladder cancer, breast cancer, cancer caused by human papilloma virus,head and neck cancer, renal cancer, and renal cell carcinoma.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        and    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein IL-2 is present at an initial concentration of about        3000 IU/mL and OKT-3 antibody is present at an initial        concentration of about 30 ng/mL in the second cell culture        medium.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a third population of TILs and is obtainableby a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first population of TILs is/has        been obtained from a tumor resected from a patient, and wherein        the second population of TILs is at least 5-fold greater in        number than the first population of TILs, and wherein the first        cell culture medium comprises IL-2; and    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        wherein IL-2 is present at an initial concentration of about        3000 IU/mL and OKT-3 antibody is present at an initial        concentration of about 30 ng/mL in the second cell culture        medium.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        and    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the rapid expansion is performed over a period not        greater than 14 days.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a third population of TILs and is obtainableby a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first population of TILs is/has        been obtained from a tumor resected from a patient, and wherein        the second population of TILs is at least 5-fold greater in        number than the first population of TILs, and wherein the first        cell culture medium comprises IL-2; and    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        wherein the rapid expansion is performed over a period not        greater than 14 days.

In embodiment, the invention provides a method of treating a cancer witha population of tumor infiltrating lymphocytes (TILs) comprising thesteps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        and    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the initial expansion is performed using a gas permeable        container.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        and    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the rapid expansion is performed using a gas permeable        container.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a third population of TILs and is obtainableby a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first population of TILs is/has        been obtained from a tumor resected from a patient, and wherein        the second population of TILs is at least 5-fold greater in        number than the first population of TILs, and wherein the first        cell culture medium comprises IL-2;    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;        wherein the initial expansion and/or the rapid expansion is        performed using a gas-permeable container.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the ratio of the second population of TILs to the        population of aAPCs in the rapid expansion is between 1 to 80        and 1 to 400.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;

(d) administering a therapeutically effective portion of the thirdpopulation of TILs to a patient with the cancer,

-   -   wherein the ratio of the second population of TILs to the        population of aAPCs in the rapid expansion is about 1 to 300.

In an embodiment, the invention provides a population of tumorinfiltrating lymphocytes (TILs) for use in treating a cancer, whereinthe population of TILs is a third population of TILs and is obtainableby a method comprising the steps:

-   -   (a) performing an initial expansion of a first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the first population of TILs is/has        been obtained from a tumor resected from a patient, and wherein        the second population of TILs is at least 5-fold greater in        number than the first population of TILs, and wherein the first        cell culture medium comprises IL-2;    -   (b) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain the        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3,        and wherein the ratio of the second population of TILs to the        population of aAPCs in the rapid expansion is between 1 to 80        and 1 to 400.

In an embodiment, the the ratio of the second population of TILs to thepopulation of aAPCs in the rapid expansion is about 1 to 300.

In an embodiment, the invention provides a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient;    -   (b) performing an initial expansion of the first population of        TILs in a first cell culture medium to obtain a second        population of TILs, wherein the second population of TILs is at        least 5-fold greater in number than the first population of        TILs, and wherein the first cell culture medium comprises IL-2;    -   (c) performing a rapid expansion of the second population of        TILs using a population of myeloid artificial antigen presenting        cells (aAPCs) in a second cell culture medium to obtain a third        population of TILs, wherein the third population of TILs is at        least 50-fold greater in number than the second population of        TILs after 7 days from the start of the rapid expansion; and        wherein the second cell culture medium comprises IL-2 and OKT-3;    -   (d) administering a therapeutically effective portion of the        third population of TILs to a patient with the cancer,    -   wherein the cancer is selected from the group consisting of        melanoma, ovarian cancer, cervical cancer, non-small-cell lung        cancer (NSCLC), lung cancer, bladder cancer, breast cancer,        cancer caused by human papilloma virus, head and neck cancer,        renal cancer, and renal cell carcinoma.

In an embodiment, the invention provides a kit for specifically inducingproliferation of a tumor infiltrating lymphocyte expressing a knownco-stimulatory molecule, the kit comprising an effective amount of anaAPC, wherein said aAPC comprises a MOLM-14 cell or a EM-3 celltransduced using a lentiviral vector (LV), wherein the LV comprises anucleic acid encoding at least one co-stimulatory ligand thatspecifically binds said known co-stimulatory molecule, wherein bindingof the known co-stimulatory molecule with said co-stimulatory ligandstimulates and expands said T cell, the kit further comprising anapplicator and an instructional material for the use of said kit.

In an embodiment, the invention provides a method for assessing thepotency of tumor infiltrating lymphocytes (TILs) comprising the stepsof:

-   -   (a) providing a plurality of mouse mastocytoma P815 cells        expressing the endogenous CD16 Fc receptor, wherein the P815        cells are transduced with a lentiviral vector based on enhanced        green fluorescent protein (GFP) and Firefly Luciferase;    -   (b) co-culturing the plurality of P815 cells TILs with and        without OKT-3 to assess T cell receptor (TCR) activation        (specific killing) or lymphokine activated killing (LAK,        non-specific killing), respectively;    -   (c) incubating for four hours;    -   (d) adding Luciferin and incubating for 5 minutes;    -   (e) reading bioluminescence intensity using a luminometer; and    -   (f) and calculating percent cytotoxicity and survival.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings.

FIG. 1 illustrates the results of rapid expansion of TILs usingirradiated allogeneic PBMC feeder cells. Each TIL line (M1015T andM1016T) (1.3×10⁵ cells) was co-cultured with 46 different irradiatedfeeders (1.3×10⁷ cells), IL-2 (3000 IU/mL) and OKT-3 (30 ng/mL) in a T25flask for 7 days. The fold expansion value for TILs was calculated onDay 7. The figure shows the number of fold expansions for two TIL linesin separate stimulation experiments, with 46 different feeder lotstested, and highlights the variability of expansion results using PBMCfeeder cells.

FIG. 2 illustrates a vector diagram of the pLV430G human 4-1BBL vector.

FIG. 3 illustrates a diagram of the 4-1BBL PCRP (polymerase chainreaction product) portion of the pLV430G human 4-1BBL vector.

FIG. 4 illustrates a vector diagram of the pLV430G human CD86 vector.

FIG. 5 illustrates a diagram of the CD86 PCRP portion of the pLV430Ghuman CD86 vector.

FIG. 6 illustrates a vector diagram of the pDONR221 human CD86 donorvector.

FIG. 7 illustrates a vector diagram of the pDONR221 human 4-1BBL donorvector.

FIG. 8 illustrates a vector diagram of the pLV430G empty vector.

FIG. 9 illustrates a vector diagram of the pDONR221 empty vector.

FIG. 10 illustrates a vector diagram of the psPAX2 helper plasmid forlentivirus production.

FIG. 11 illustrates a vector diagram of the pCIGO-VSV.G helper plasmidfor lentivirus production.

FIG. 12 illustrates the results of flow cytometry experiments on MOLM-14cells before lentiviral transfection (“Untransfected”) and aftertransfection (“Transfected”), confirming the expression of CD137 andCD86 on engineered MOLM-14 cells.

FIG. 13 illustrates the results of rapid expansion of TILs usingirradiated parental unmodified MOLM-14 cells (“Parent MOLM14”),engineered MOLM-14 cells (CD86/4-1BBL, “Engineered MOLM14”), or PBMCfeeders (“Feeders”) for TIL lot M1032-T2. TIL were co-cultured with PBMCfeeders or parental or engineered MOLM14 cells at 1:100 ratios withOKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split onDay 6 and 11. Each dot represents cell numbers determined on Day 0, 6,11 and 14 respectively. A logarithmic scale is used.

FIG. 14 illustrates results as shown in FIG. 13, depicted using a linearscale.

FIG. 15 illustrates results for TIL lot M1033-T6 with other parametersas given in FIG. 13, using a logarithmic scale.

FIG. 16 illustrates results as shown in FIG. 14, depicted using a linearscale.

FIG. 17 illustrates the results of rapid expansions of TILs usingengineered MOLM-14 cells expressing CD86 and 4-1BBL (“TIL+EngineeredMOLM14 (CD86/41BB)+OKT3”) or irradiated PBMC feeders(“TIL+Feeders+OKT3”). TIL were co-cultured with PBMC feeders orengineered MOLM-14 cells (aMOLM14) at 1:100 ratios plus OKT-3 (30 ng/mL)and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11.Each point represents cell numbers determined on Day 14.

FIG. 18 illustrates the results of experiments in which TILs (2×10⁴)were cultured with different ratios (1:10, 1:30, and 1:100, denoted“10”, “30”, and “100”, respectively) of parental MOLM-14 (“MOLM14”)cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”),or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000IU/mL) in wells of a 24-well G-Rex plate. A control was performed usingonly OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC-”). Each conditionwas cultured in triplicate. Cultures were fed with fresh media and IL-2on Day 4 and 7. Viable cells were counted on Day 7. The bar graphrepresented here shows the mean plus standard deviation (SD) of viablecell numbers counted on Day 11. The p-value was calculated by thestudent ‘t’ test.

FIG. 19 illustrates the results of TILs cultured with different ratios(1:30, 1:100, and 1:300, denoted “30”, “100”, and “300”, respectively)of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3(30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex cultureplates. Viable cells were counted on day 11 and plotted. Otherconditions are as in FIG. 18.

FIG. 20 illustrates the results of TILs cultured with different ratios(1:50, 1:100, and 1:200, denoted “50”, “100”, and “200”, respectively)of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3(30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex cultureplates. Cells were counted on day 14. Other conditions are as in FIG.18.

FIG. 21 illustrates the results of TILs cultured with different ratios(1:100, 1:200, 1:400, and 1:800, denoted “100”, “200”, “400”, and “800”,respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells(“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL(“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in thesingle 24 well G-Rex culture plates. Cells were counted on day 14. Otherconditions are as in FIG. 18.

FIG. 22 illustrates a sunburst visualization showing fine distributionof Live, T cell receptor (TCR) α/β, CD4, CD8, CD27, CD28, and CD57 TILsexpanded with PBMC feeders.

FIG. 23 illustrates a sunburst visualization showing fine distributionof Live, TCR α/β, CD4, CD8, CD27, CD28, and CD57 TILs expanded withaMOLM14 aAPCs.

FIG. 24 depicts a flow cytometry contour plot showing memory subset(CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4⁺, or CD8⁺ TILs.

FIG. 25 illustrates phenotypic characterization of the T cell subset,CD4⁺ and CD8⁺ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3⁺cells using a SPADE tree. The color gradient is proportional to the meanfluorescence intensity (MFI) of LAG3, PD1, and CD137.

FIG. 26 illustrates phenotypic characterization of the T cell subset,CD4⁺ and CD8⁺ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3⁺cells using a SPADE tree. The color gradient is proportional to the MFICD69, CD154, KLRG1, and TIGIT

FIG. 27 illustrates oxygen consumption rate (OCR) of TIL after expansionwith Feeders or aMOLM14 measured during a mitochondrial stress test.Each data point represents mean±standard error of the mean (SEM)measured in triplicate.

FIG. 28 illustrates extracellular acidification rate (ECAR) of TIL afterexpansion with Feeders or aMOLM14 measured during a mitochondrial stresstest. Each data point represents mean±SEM measured in triplicate.

FIG. 29 illustrates a vector diagram of the destination vector pLV4301G.

FIG. 30 illustrates a vector diagram of donor vector 1, pMK 7c12 antimFC scFv CoOp ECORV SacII L1R5.

FIG. 31 illustrates a vector diagram of donor vector 2, pMK hCD8ascaffold TN L5 L2.

FIG. 32 illustrates a vector diagram of final vector used for lentiviralproduction, pLV4301G 7C12 scFv mIgG hCD8 flag.

FIG. 33 illustrates a vector diagram of the destination vector pLV4301G.

FIG. 34 illustrates a vector diagram of donor vector 1, pMK 8B3 anti mFCscFv CoOp ECORV SacII L1R5.

FIG. 35 illustrates a vector diagram of donor vector 2, pMK hCD8ascaffold TN L5 L2.

FIG. 36 illustrates a vector diagram of final vector used for lentiviralproduction, pLV4301G 8B3 scFv mIgG hCD8 flag.

FIG. 37 illustrates the results of flow cytometry experiments on EM-3cells before lentiviral transfection (“Untransfected”) and aftertransfection (“Transfected”), confirming the expression of CD137 andCD86 on engineered EM-3 cells.

FIG. 38 illustrates the results of experiments wherein TILs wereco-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.

FIG. 39 illustrates the results of experiments wherein TILs wereco-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.

FIG. 40 illustrates the results of experiments wherein TILs wereco-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000IU/mL), with or without OKT-3 (30 ng/mL). The bar graph shows cellnumbers determined on Day 11.

FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs atdifferent TIL:aAPC ratios.

FIG. 42 illustrates the results of TIL expansions with EM-3 aAPCs. TILs(2×10⁴) were co-cultured with five different PBMC feeder lots or aEM3(in triplicate) at a 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24well plate. Viable cells were counted on Day 14. The graph shows viablecell numbers (mean) with 95% confidence interval counted on Day 14.

FIG. 43 illustrates the results of TIL expansions with EM-3 aAPCs andMOLM-14 aAPCs. TILs (2×10⁴) were co-cultured with five different PBMCfeeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. The graphshows viable cell numbers (mean) with 95% confidence interval counted onDay 14.

FIG. 44 illustrates a sunburst visualization to show fine distributionof Live, TCR α/β, CD4⁺, and CD8⁺ TILs expanded with aEM3 aAPCs or PBMCfeeders (TIL batch M1054).

FIG. 45 illustrates the sunburst visualization to show fine distributionof Live, TCR α/β, CD4⁺, and CD8⁺ TILs expanded with aEM3 aAPCs or PBMCfeeders (TIL batch M1055).

FIG. 46 illustrates the CD4⁺ and CD8⁺ SPADE tree of TILs expanded withaEM3 aAPCs or PBMC feeders using CD3⁺ cells. The color gradient isproportional to the MFI of LAG-3, TIM-3, PD-1, and CD137.

FIG. 47 illustrates the CD4⁺ and CD8⁺ SPADE tree of TILs expanded withaEM3 aAPCs or PBMC feeders using CD3⁺ cells. The color gradient isproportional to the MFI of CD69, CD154, KLRG1, and TIGIT.

FIG. 48 illustrates a summary of spare respiratory capacity measured bythe Seahorse XF Mito stress test.

FIG. 49 illustrates a summary of glycolytic reserve measured by theSeahorse XF Mito stress test.

FIG. 50 illustrates a mitochondrial stain of live TILs expanded againstPBMC or aEM3 using MitoTracker dye, which stains mitochondria in livecells and for which accumulation is dependent upon membrane potential.TILs expanded against PBMC or aEM3 were stained L/D Aqua followed byMitoTracker red dye. Data shown are MitoTracker positive (MFI) cellsgated on live population.

FIG. 51 illustrates results of a P815 BRLA for cytotoxic potency andfunctional activity, comparing TILs expanded with PBMC feeders to TILsexpanded using aMOLM14 aAPCs.

FIG. 52 illustrates results of a P815 BRLA for cytotoxic potency andfunctional activity, comparing TILs expanded with PBMC feeders to TILsexpanded using aEM3 aAPCs.

FIG. 53 illustrates IFN-γ release for two batches of TILs followingovernight stimulation (“S”) with microbeads coated withanti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparingTILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. *p<0.05, ** p<0.005, *** p<0.001, ns=not significant.

FIG. 54 illustrates IFN-γ release for three batches of TILs followingovernight stimulation (“S”) with microbeads coated withanti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparingTILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. *p<0.05, ** p<0.005, *** p<0.001, ns=not significant.

FIG. 55 illustrates Granzyme B release for two batches of TILs followingovernight stimulation (“S”) with microbeads coated withanti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparingTILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. *p<0.05, ** p<0.005, *** p<0.001, ns=not significant.

FIG. 56 illustrates Granzyme B release for three batches of TILsfollowing overnight stimulation (“S”) with microbeads coated withanti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparingTILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. *p<0.05, ** p<0.005, *** p<0.001, ns=not significant.

FIG. 57 illustrates a TIL expansion and treatment process. aAPCs of thepresent invention may be used in both the pre-REP stage (top half offigure) or REP stage (bottom half of figure) and may be added when IL-2is added to each cell culture. Step 1 refers to the addition of 4 tumorfragments into 10 G-Rex 10 flasks. At step 2, approximately 40×10⁶ TILsor greater are obtained. At step 3, a split occurs into 36 G-Rex 100flasks for REP. TILs are harvested by centrifugation at step 4. FreshTIL product is obtained at step 5 after a total process time ofapproximate 43 days, at which point TILs may be infused into a patient.

FIG. 58 illustrates a treatment protocol for use with TILs expanded withaAPCs. Surgery (and tumor resection) occurs at the start, andlymphodepletion chemo refers to non-myeloablative lymphodepletion withchemotherapy as described elsewhere herein.

FIG. 59 illustrates Bioluminescent Redirected Lysis Assay (BRLA)results, showing percentage cytotoxicity of TIL batch M1033T-1 whenco-cultured with P815 Clone G6 (with and without anti-CD3) at individualeffector:target ratios.

FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) datashowing amount of IFN-γ released against different ratios of effector totarget cells.

FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 whenco-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of1:1 effector to target cells for 4 hr and 24 hr co-culture.

FIG. 62 illustrates BRLA results for TIL batch M1030. Cytotoxicity(measured as LU₅₀/1×10⁶ TIL) by BRLA is 26±16.

FIG. 63 illustrates standard chromium release assay for TIL batch M1030.Cytotoxicity (measured as LU₅₀/1×10⁶ TIL) by the chromium release assayis 22.

FIG. 64 illustrates BRLA results for TIL batch M1053, showing the lyticunits of the TILs by BRLA as 70±17.

FIG. 65 illustrates standard chromium release assay results for TILbatch M1053, also showing lytic unit of the TILs by chromium assay as14±5. Comparison of this result with FIG. 64 shows the comparableperformance of the BRLA and chromium release assay.

FIG. 66 illustrates the linear relationship between IFN-γ release andcytotoxic potential of TILs.

FIG. 67 illustrates ELISpot results for IFN-γ.

FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053.

FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030.

FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053Tand M1030T. This data confirms the potency of the TILs shown by theBRLA.

FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053.

FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030.

FIG. 73 illustrates ELISpot data showing TNF-α release by M1053T andM1030T. This data confirms the potency of the TILs shown by the BRLA.

FIG. 74 illustrates enzymatic TNF-α release for TIL batch M1053.

FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030.

FIG. 76 illustrates changes in cell populations of aEM3 cells (C712 (A)and 8B5 (B)) when weaning such cell populations off of FBS to hAB serummedia.

FIG. 77 illustrates changes in cell populations of duringfreeze-thaw-recovery cycles with aEM3 cell populations suspended invarious freezing media.

FIG. 78 illustrates the growth of aEM3 cells in gas permeable cellculture flasks over an eight-day time course.

FIG. 79 illustrates a flow panel analysis to determine the purity ofaEM3 cells.

FIG. 80 illustrates the results of a flow panel analysis used todetermine the purity of aEM3 cells.

FIG. 81 illustrates the differences in cytokine expression between aEM3feeder cells and PBMC feeders stimulated by OKT3.

FIG. 82 illustrates that TIL may advantageously expanded (pre-REP) withserum free media (i.e., CTS Optmizer) to provide increased cell numbersas compared to CM1.

FIG. 83 and FIG. 84 illustrate that TIL may advantageously expanded withserum free media (i.e., CTS Optmizer) to provide increased cell numbersas compared to CM1 at Day 11 (PreREP) (FIG. 83) and Day 22 (Pre- andPost-REP) (FIG. 84).

FIG. 85 illustrates that aAPC cells (i.e., aEM3 cells) can be grown andusing serum free media. Specifically, CTS OpTimizer and Prime-TCDM werefound to be effective in growing aEM3 as compared to cDMEM (10% hSerum).Data shown were mean±SD of five separate experiments. The p value wascalculated by the student t-test. *P<0.05.

FIG. 86 and FIG. 87 illustrate the results of two experiments thatdemonstrate the rapid recovery of aEM3 cells from the TIL-R3 cell lineon day 3 following cryopreservation. FIG. 86 illustrates the total cellcounts for experiment one and FIG. 87 illustrates the total cell countsfor experiment two.

FIG. 88 illustrates the growth of aEM3 cells from the TIL-R3 cell linefollowing cryopreservation where the cells were plated and grown for 9days. Cell counts were measured every three days post thaw.

FIG. 89 illustrates the growth of aEM3 cells from the TIL-R3 cell linefollowing cryopreservation where the cells were plated in GREX 10 flasksand grown for 8 days. Cell counts were measured every four days postthaw.

FIG. 90 illustrates a vector diagram of the pLenti-C-Myc-DDK human OX40Lvector.

FIG. 91 illustrates the results of flow cytometry analysis of TILsexpanded in a REP with the aEM3 cell line and PBMC feeders, showing thatTILs cultured with aEM3 promotes CD8⁺ TIL skewness.

FIG. 92 illustrates the numbers of viable cells obtained fromexperiments wherein TILs were expanded in a REP with the aEM3 cell lineand PBMC feeders.

FIG. 93 illustrates the numbers of CD3⁺ cells obtained from experimentswherein TILs were expanded in a REP with the aEM3 cell line and PBMCfeeders.

FIG. 94 illustrates the numbers of CD3⁻ cells obtained from experimentswherein TILs were expanded in a REP with the aEM3 cell line and PBMCfeeders.

FIG. 95 illustrates the results of telomere length analysis using a qPCRmethod.

FIG. 96 illustrates a schematic diagram of an embodiment of an aAPC ofthe present invention.

FIG. 97 illustrates a schematic diagram of an embodiment of an aAPC ofthe present invention.

FIG. 98 illustrates a schematic diagram of an embodiment of an aAPC ofthe present invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is an amino acid sequence for the heavy chain of muromonab.

SEQ ID NO:2 is an amino acid sequence for the light chain of muromonab.

SEQ ID NO:3 is an amino acid sequence for recombinant human IL-2.

SEQ ID NO:4 is an amino acid sequence for aldesleukin.

SEQ ID NO:5 is an amino acid sequence for recombinant human IL-7.

SEQ ID NO:6 is an amino acid sequence for recombinant human IL-15.

SEQ ID NO:7 is an amino acid sequence for recombinant IL-21.

SEQ ID NO:8 is the amino acid sequence of human CD86.

SEQ ID NO:9 is the amino acid sequence of human 4-1BBL (CD137L).

SEQ ID NO:10 is the amino acid sequence of human OX40L (CD134L).

SEQ ID NO:11 is the amino acid sequence of human CD28.

SEQ ID NO:12 is the amino acid sequence of human CTLA-4.

SEQ ID NO:13 is the amino acid sequence of human 4-1BB (CD137).

SEQ ID NO:14 is the amino acid sequence of human OX40 (CD134).

SEQ ID NO:15 is a nucleotide sequence for the pLV430G 4-1BBL emptyvector.

SEQ ID NO:16 is a nucleotide sequence for the 4-1BBL CoOP portion of thepLV430G human 4-1BBL vector.

SEQ ID NO:17 is a nucleotide sequence for the 4-1BBL PCRP.

SEQ ID NO:18 is a nucleotide sequence for the pLV430G hCD86 emptyvector.

SEQ ID NO:19 is a nucleotide sequence for the hCD86 CoOP portion of thepLV430G human hCD86 vector.

SEQ ID NO:20 is a nucleotide sequence for the hCD86 CoOP B1 B2 PCRPportion of the pLV430G human hCD86 vector.

SEQ ID NO:21 is a nucleotide sequence for the pDONR221 hCD86 vector.

SEQ ID NO:22 is a nucleotide sequence for the pDONR221 4-1BBL vector.

SEQ ID NO:23 is a nucleotide sequence for the pLV430G vector.

SEQ ID NO:24 is a nucleotide sequence for the pDONR221 vector.

SEQ ID NO:25 is a nucleotide sequence for the psPAX2 helper plasmid forlentiviral production.

SEQ ID NO:26 is a nucleotide sequence for the pCIGO-VSV.G helper plasmidfor lentiviral production.

SEQ ID NO:27 is the amino acid sequence of the mFc-7C12 scFv clone.

SEQ ID NO:28 is the amino acid sequence of the mFc-8B3 scFv clone.

SEQ ID NO:29 is a nucleotide sequence for the mFC-7C12 scFv.

SEQ ID NO:30 is a nucleotide sequence for the mFC-8B3 scFv.

SEQ ID NO:31 is a nucleotide sequence for the destination vectorpLV4301G.

SEQ ID NO:32 is a nucleotide sequence for the donor vector 1, pMK 7c12anti mFC scFv CoOp ECORV SacII L1R5.

SEQ ID NO:33 is a nucleotide sequence for the donor vector 2, pMK hCD8ascaffold TN L5 L2.

SEQ ID NO:34 is a nucleotide sequence for the final vector used forlentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.

SEQ ID NO:35 is a nucleotide sequence for the destination vector,pLV4301G.

SEQ ID NO:36 is a nucleotide sequence for the donor vector 1, pMK 8B3anti mFC scFv CoOp ECORV SacII L1R5.

SEQ ID NO:37 is a nucleotide sequence for the donor vector 2, pMK hCD8ascaffold TN L5 L2.

SEQ ID NO:38 is a nucleotide sequence for the final vector used forlentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.

SEQ ID NO:39 is a nucleotide sequence for pLenti-C-Myc-DDK OX40L vectorfor lentiviral production.

SEQ ID NO:40 is a nucleotide sequence for Tel-1b primer used forquantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:41 is a nucleotide sequence for Tel-2b primer used forquantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:42 is a nucleotide sequence for Tel-1b primer used forquantitative polymerase chain reaction measurements of telomere length.

SEQ ID NO:43 is a nucleotide sequence for Tel-1b primer used forquantitative polymerase chain reaction measurements of telomere length.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

Definitions

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients to a human subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the human subject at the same time. Co-administration includessimultaneous administration in separate compositions, administration atdifferent times in separate compositions, or administration in acomposition in which two or more active pharmaceutical ingredients arepresent. Simultaneous administration in separate compositions andadministration in a composition in which both agents are present is alsoencompassed in the methods of the invention.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body. Aptly, the cell, tissue and/or organ maybe returned to the subject's body in a method of surgery or treatment.

The term “antigen” refers to a substance that induces an immuneresponse. In some embodiments, an antigen is a molecule capable of beingbound by an antibody or a T cell receptor (TCR) if presented by majorhistocompatibility complex (MEW) molecules. The term “antigen”, as usedherein, also encompasses T cell epitopes. An antigen is additionallycapable of being recognized by the immune system. In some embodiments,an antigen is capable of inducing a humoral immune response or acellular immune response leading to the activation of B lymphocytesand/or T lymphocytes. In some cases, this may require that the antigencontains or is linked to a Th cell epitope. An antigen can also have oneor more epitopes (e.g., B- and T-epitopes). In some embodiments, anantigen will preferably react, typically in a highly specific andselective manner, with its corresponding antibody or TCR and not withthe multitude of other antibodies or TCRs which may be induced by otherantigens.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the human subject and disease condition being treated (e.g.,the weight, age and gender of the subject), the severity of the diseasecondition, the manner of administration, etc. which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

A “therapeutic effect” as that term is used herein, encompasses atherapeutic benefit and/or a prophylactic benefit in a human subject. Aprophylactic effect includes delaying or eliminating the appearance of adisease or condition, delaying or eliminating the onset of symptoms of adisease or condition, slowing, halting, or reversing the progression ofa disease or condition, or any combination thereof.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” is intended to include any and all solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and inert ingredients. The use of suchpharmaceutically acceptable carriers or pharmaceutically acceptableexcipients for active pharmaceutical ingredients is well known in theart. Except insofar as any conventional pharmaceutically acceptablecarrier or pharmaceutically acceptable excipient is incompatible withthe active pharmaceutical ingredient, its use in the therapeuticcompositions of the invention is contemplated. Additional activepharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are described herein.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to herein as “freshly harvested” or“a first population of TILs”), and “secondary TILs” are any TIL cellpopulations that have been expanded or proliferated as discussed herein,including, but not limited to bulk TILs and expanded TILs (“REP TILs” or“post-REP TILs”, or “second population of TILs” or “third population ofTILs” where appropriate).

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient.

By “cryopreserved TILs” herein is meant that TILs are treated and storedin the range of about −150° C. to −60° C. General methods forcryopreservation are also described elsewhere herein, including in theExamples. For clarity, “cryopreserved TILs” are distinguishable fromfrozen tissue samples which may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits.

The term “central memory T cell” refers to a subset of T cells that inthe human are CD45R0+ and constitutively express CCR7 (CCR7^(hi)) andCD62L (CD62^(hi)). The surface phenotype of central memory T cells alsoincludes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors forcentral memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Centralmemory T cells primarily secret IL-2 and CD40L as effector moleculesafter TCR triggering. Central memory T cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human ormammalian T cells that, like central memory T cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7^(lo)) and areheterogeneous or low for CD62L expression (CD62L^(lo)). The surfacephenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T cellsinclude BLIMP1. Effector memory T cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T cells carrylarge amounts of perforin.

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” in the context of two or more nucleic acids or polypeptides,refer to two or more sequences or subsequences that are the same or havea specified percentage of nucleotides or amino acid residues that arethe same, when compared and aligned (introducing gaps, if necessary) formaximum correspondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

The term “conservative amino acid substitutions” means amino acidsequence modifications which do not abrogate the binding of an antibodyto an antigen or a protein to its ligand. Conservative amino acidsubstitutions include the substitution of an amino acid in one class byan amino acid of the same class, where a class is defined by commonphysicochemical amino acid side chain properties and high substitutionfrequencies in homologous proteins found in nature, as determined, forexample, by a standard Dayhoff frequency exchange matrix or BLOSUMmatrix. Six general classes of amino acid side chains have beencategorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala,Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V(Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,substitution of an Asp for another class III residue such as Asn, Gln,or Glu, is a conservative substitution. Thus, a predicted nonessentialamino acid residue in a 4-1BBL or CD86 protein is preferably replacedwith another amino acid residue from the same class. Methods ofidentifying amino acid conservative substitutions which do not eliminateantigen or ligand binding are well-known in the art (see, e.g.,Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al.,Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl.Acad. Sci. USA 1997, 94, 412-417).

The term “retrovirus” refers to RNA viruses that utilize reversetranscriptase during their replication cycle, wherein retroviral genomicRNA is converted into double-stranded DNA by reverse transcriptase. Thedouble-stranded DNA form is integrated into the chromosome of theinfected cell (a “provirus”). The provirus serves as a template for RNApolymerase II and directs the expression of RNA molecules which encodethe structural proteins and enzymes needed to produce new viralparticles. At each end of the provirus are structures called “longterminal repeats” or “LTRs.” The LTR contains numerous regulatorysignals including transcriptional control elements, polyadenylationsignals and sequences needed for replication and integration of theviral genome. Several genera included within the family Retroviridae,including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C,Oncovirus D, Lentivirus, Gammaretrovirus, and Spumavirus. Some of theretroviruses are oncogenic (i.e., tumorigenic), while others are not.The oncoviruses induce sarcomas, leukemias, lymphomas, and mammarycarcinomas in susceptible species. Retroviruses infect a wide variety ofspecies, and may be transmitted both horizontally and vertically.Because they are integrated into the host DNA, they are capable oftransmitting sequences of host DNA from cell to cell. Examplegammaretroviral vectors include those derived from the amphotropicMoloney murine leukemia virus (MLV-A), which use cell surface phosphatetransporter receptors for entry and then permanently integrate intoproliferating cell chromosomes. The amphotropic MLV vector system hasbeen well established and is a popular tool for gene delivery (See,e.g., Gordon and Anderson, Curr. Op. Biotechnol., 1994, 5, 611-616 andMiller, et al., Meth. Enzymol., 1993, 217, 581-599, the disclosures ofwhich are incorporated herein by reference.

The term “lentivirus” refers to a genus that includes HIV (humanimmunodeficiency virus; including HIV type 1, and HIV type 2),visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) insheep, the caprine arthritis-encephalitis virus, which causes immunedeficiency, arthritis, and encephalopathy in goats; equine infectiousanemia virus, which causes autoimmune hemolytic anemia, andencephalopathy in horses; feline immunodeficiency virus (Hy), whichcauses immune deficiency in cats; bovine immune deficiency virus (BIV),which causes lymphadenopathy, lymphocytosis, and possibly centralnervous system infection in cattle; and simian immunodeficiency virus(SIV), which cause immune deficiency and encephalopathy in sub-humanprimates. Diseases caused by these viruses are characterized by a longincubation period and protracted course. Usually, the viruses latentlyinfect monocytes and macrophages, from which they spread to other cells.HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in the Tcell antigen receptor of mature T cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab. Anti-CD3 antibodies also include theUHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or variant thereof, including human, humanized,chimeric, or murine antibodies, directed against the CD3 receptor in theT cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706.

TABLE 1 Amino acid sequences of muromonab. Identifier (Description)Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60(Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120chain) KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60(Muromonab lightFRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120chain) SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4,902,502, the disclosures of which are incorporated by referenceherein. Formulations of IL-2 suitable for use in the invention aredescribed in U.S. Pat. No. 6,706,289, the disclosure of which isincorporated by reference herein.

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-7 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:5).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:6).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4⁺ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:7).

TABLE 2 Amino acid sequences of interleukins. Identifier (Description)Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL 60(recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2 RWITFCQSII STLT 134 (rhIL-2)) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE 60(aldesleukin)ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT 132 SEQ ID NO: 5MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA 60(recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 (rhIL-7))SEQ ID NO: 6MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI 60(recombinantHDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS 115human IL-15 (rhIL-15)) SEQ ID NO: 7MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60(recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21 HLSSRTHGSE DS 132 (rhIL-21))

The term “myeloid cell” as used herein refers to cells of the myeloidlineage or derived therefrom. The myeloid lineage includes a number ofmorphologically, phenotypically, and functionally distinct cell typesincluding different subsets of granulocytes (neutrophils, eosinophils,and basophils), monocytes, macrophages, erythrocytes, megakaryocytes,and mast cells. In certain embodiments, the cell is a cell derived froma cell line of myeloid lineage.

“MOLM-14” refers to a human leukemia cell line which was establishedfrom the peripheral blood of a patient with relapsed acute monocyticleukemia, and initial phenotypic characterization indicated the presenceof at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15,CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155.Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypiccharacterization of MOLM-14 found higher levels of HLA-AB/C, CD64, CD80,ICOS-L, CD58, and lower levels of CD86. The MOLM-14 cell line isdeposited at DSMZ under Accession No. ACC777. The closely relatedMOLM-13 cell line is deposited at DSMZ under Accession No. ACC554. Asused herein the term “MOLM-14 cell” refers to a MOLM-14 cell and/or acell derived from the deposited MOLM-14 parental cell line. As usedherein the term “MOLM-13 cell” refers to a MOLM-13 cell and/or a cellderived from the deposited MOLM-13 parental cell line.

“EM-3” refers to a human cell line was established from the bone marrowof a patient with Philadelphia chromosome-positive CML. Konopka, et al.,Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. Phenotypic characterizationfor EM-3 cells indicates the presence of at least the following markers:CD13, CD15, and CD33. The EM-3 cell line is deposited at DSMZ underAccession No. ACC134 whilst the closely related EM-2 cell line isdeposited at DSMZ under Accession No. ACC135. As used herein the term“EM-3 cell” refers to a EM-3 cell and/or a cell derived from thedeposited EM-3 parental cell line.

As used herein, the term “a CD86 protein” may refer to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:8 or aprotein comprising an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence depicted in SEQ ID NO:8, e.g., 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term “4-1BBL” or “CD137L” may refer to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:9 or aprotein comprising an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence depicted in SEQ ID NO:9, e.g., 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

As used herein, the term “OX40L” or “CD137L” may refer to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:10 or aprotein comprising an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence depicted in SEQ ID NO:10, e.g., 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

The term “biosimilar” means a biological product, including a monoclonalantibody or fusion protein, that is highly similar to a U.S. licensedreference biological product notwithstanding minor differences inclinically inactive components, and for which there are no clinicallymeaningful differences between the biological product and the referenceproduct in terms of the safety, purity, and potency of the product.Furthermore, a similar biological or “biosimilar” medicine is abiological medicine that is similar to another biological medicine thathas already been authorized for use by the European Medicines Agency.The term “biosimilar” is also used synonymously by other national andregional regulatory agencies. Biological products or biologicalmedicines are medicines that are made by or derived from a biologicalsource, such as a bacterium or yeast. They can consist of relativelysmall molecules such as human insulin or erythropoietin, or complexmolecules such as monoclonal antibodies. For example, if the referenceIL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drugregulatory authorities with reference to aldesleukin is a “biosimilarto” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe,a similar biological or “biosimilar” medicine is a biological medicinethat is similar to another biological medicine that has already beenauthorized for use by the European Medicines Agency (EMA). The relevantlegal basis for similar biological applications in Europe is Article 6of Regulation (EC) No 726/2004 and Article 10(4) of Directive2001/83/EC, as amended and therefore in Europe, the biosimilar may beauthorized, approved for authorization or subject of an application forauthorization under Article 6 of Regulation (EC) No 726/2004 and Article10(4) of Directive 2001/83/EC. The already authorized originalbiological medicinal product may be referred to as a “referencemedicinal product” in Europe. Some of the requirements for a product tobe considered a biosimilar are outlined in the CHIMP Guideline onSimilar Biological Medicinal Products. In addition, product specificguidelines, including guidelines relating to monoclonal antibodybiosimilars, are provided on a product-by-product basis by the EMA andpublished on its website. A biosimilar as described herein may besimilar to the reference medicinal product by way of qualitycharacteristics, biological activity, mechanism of action, safetyprofiles and/or efficacy. In addition, the biosimilar may be used or beintended for use to treat the same conditions as the reference medicinalproduct. Thus, a biosimilar as described herein may be deemed to havesimilar or highly similar quality characteristics to a referencemedicinal product. Alternatively, or in addition, a biosimilar asdescribed herein may be deemed to have similar or highly similarbiological activity to a reference medicinal product. Alternatively, orin addition, a biosimilar as described herein may be deemed to have asimilar or highly similar safety profile to a reference medicinalproduct. Alternatively, or in addition, a biosimilar as described hereinmay be deemed to have similar or highly similar efficacy to a referencemedicinal product. As described herein, a biosimilar in Europe iscompared to a reference medicinal product which has been authorized bythe EMA. However, in some instances, the biosimilar may be compared to abiological medicinal product which has been authorized outside theEuropean Economic Area (a non-EEA authorized “comparator”) in certainstudies. Such studies include for example certain clinical and in vivonon-clinical studies. As used herein, the term “biosimilar” also relatesto a biological medicinal product which has been or may be compared to anon-EEA authorized comparator. Certain biosimilars are proteins such asantibodies, antibody fragments (for example, antigen binding portions)and fusion proteins. A protein biosimilar may have an amino acidsequence that has minor modifications in the amino acid structure(including for example deletions, additions, and/or substitutions ofamino acids) which do not significantly affect the function of thepolypeptide. The biosimilar may comprise an amino acid sequence having asequence identity of 97% or greater to the amino acid sequence of itsreference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilarmay comprise one or more post-translational modifications, for example,although not limited to, glycosylation, oxidation, deamidation, and/ortruncation which is/are different to the post-translationalmodifications of the reference medicinal product, provided that thedifferences do not result in a change in safety and/or efficacy of themedicinal product. The biosimilar may have an identical or differentglycosylation pattern to the reference medicinal product. Particularly,although not exclusively, the biosimilar may have a differentglycosylation pattern if the differences address or are intended toaddress safety concerns associated with the reference medicinal product.Additionally, the biosimilar may deviate from the reference medicinalproduct in for example its strength, pharmaceutical form, formulation,excipients and/or presentation, providing safety and efficacy of themedicinal product is not compromised. The biosimilar may comprisedifferences in for example pharmacokinetic (PK) and/or pharmacodynamic(PD) profiles as compared to the reference medicinal product but isstill deemed sufficiently similar to the reference medicinal product asto be authorized or considered suitable for authorization. In certaincircumstances, the biosimilar exhibits different binding characteristicsas compared to the reference medicinal product, wherein the differentbinding characteristics are considered by a Regulatory Authority such asthe EMA not to be a barrier for authorization as a similar biologicalproduct. The term “biosimilar” is also used synonymously by othernational and regional regulatory agencies.

As used herein, the term “variant” encompasses but is not limited toproteins, antibodies or fusion proteins which comprise an amino acidsequence which differs from the amino acid sequence of a referenceprotein or antibody by way of one or more substitutions, deletionsand/or additions at certain positions within or adjacent to the aminoacid sequence of the reference protein or antibody. The variant maycomprise one or more conservative substitutions in its amino acidsequence as compared to the amino acid sequence of a reference proteinor antibody. Conservative substitutions may involve, e.g., thesubstitution of similarly charged or uncharged amino acids. The variantretains the ability to specifically bind to the antigen of the referenceprotein or antibody. The term “variant” also includes pegylatedantibodies or proteins.

“Pegylation” refers to a modified antibody, or a fragment thereof, orprotein that typically is reacted with polyethylene glycol (PEG), suchas a reactive ester or aldehyde derivative of PEG, under conditions inwhich one or more PEG groups become attached to the antibody, antibodyfragment, or protein. Pegylation may, for example, increase thebiological (e.g., serum) half life of the antibody or protein.Preferably, the pegylation is carried out via an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C₁-C₁₀) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. Theantibody or protein to be pegylated may be an aglycosylated antibody.Methods for pegylation are known in the art and can be applied to theantibodies and proteins described herein, as described for example inEuropean Patent Nos. EP 0154316 and EP 0401384.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

Artificial Antigen Presenting Cells

In an embodiment, the invention includes an isolated artificial antigenpresenting cell (aAPC) comprising a cell that expresses HLA-AB/C, CD64,CD80, ICOS-L, and CD58, and is modified to express one or morecostimulatory molecules. In an embodiment, the invention includes anaAPC comprising a MOLM-14 cell that is modified to express one or morecostimulatory molecules. In an embodiment, the invention includes anaAPC comprising a MOLM-13 cell that is modified to express one or morecostimulatory molecules.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell that endogenously expresses HLA-AB/C, CD64, CD80, ICOS-L, and CD58,wherein the cell is modified to express a CD86 protein comprising anamino acid sequence as set forth in SEQ ID NO:8, and conservative aminoacid substitutions thereof, and a 4-1BBL protein comprising an aminoacid sequence as set forth in SEQ ID NO:9, and conservative amino acidsubstitutions thereof, and wherein the CD86 protein and the 4-1BBLprotein are expressed on the surface of the MOLM-14 cell.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-14 cell expresses CD86 and 4-1BBL.In an embodiment, the invention includes an aAPC comprising a MOLM-13cell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-13 cell expresses CD86 and 4-1BBL.In an embodiment, the invention includes a method of preparing any ofthe foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a CD86 protein comprising an amino acidsequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising anamino acid sequence as set forth in SEQ ID NO:9, wherein the CD86protein and the 4-1BBL protein are expressed on the surface of theMOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a CD86 protein comprisingan amino acid sequence as set forth in SEQ ID NO:8, and conservativeamino acid substitutions thereof, and a 4-1BBL protein comprising anamino acid sequence as set forth in SEQ ID NO:9, and conservative aminoacid substitutions thereof, wherein the CD86 protein and the 4-1BBLprotein are expressed on the surface of the MOLM-13 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a CD86 protein comprising a sequence withgreater than 99% identity to an amino acid sequence as set forth in SEQID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe MOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-14 cell modified to express a CD86 protein comprisinga sequence with greater than 98% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the MOLM-14 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-14 cell modified to express a CD86protein comprising a sequence with greater than 97% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the MOLM-14 cell. In anembodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a CD86 protein comprising a sequence with greaterthan 96% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 96%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe MOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-14 cell modified to express a CD86 protein comprisinga sequence with greater than 95% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the MOLM-14 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-14 cell modified to express a CD86protein comprising a sequence with greater than 90% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the MOLM-14 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13cell modified to express a CD86 protein comprising an amino acidsequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising anamino acid sequence as set forth in SEQ ID NO:9, wherein the CD86protein and the 4-1BBL protein are expressed on the surface of theMOLM-13 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a CD86 protein comprisingan amino acid sequence as set forth in SEQ ID NO:8, and conservativeamino acid substitutions thereof, and a 4-1BBL protein comprising anamino acid sequence as set forth in SEQ ID NO:9, and conservative aminoacid substitutions thereof, wherein the CD86 protein and the 4-1BBLprotein are expressed on the surface of the MOLM-13 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13cell modified to express a CD86 protein comprising a sequence withgreater than 99% identity to an amino acid sequence as set forth in SEQID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe MOLM-13 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a CD86 protein comprisinga sequence with greater than 98% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the MOLM-13 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-13 cell modified to express a CD86protein comprising a sequence with greater than 97% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the MOLM-13 cell. In anembodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a CD86 protein comprising a sequence with greaterthan 96% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 96%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe MOLM-13 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a CD86 protein comprisinga sequence with greater than 95% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the MOLM-13 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-13 cell modified to express a CD86protein comprising a sequence with greater than 90% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the MOLM-13 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding OX40L, and wherein the MOLM-14 cell expresses CD86 and OX40L.In an embodiment, the invention includes an aAPC comprising a MOLM-13cell transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding OX40L, and wherein the MOLM-13 cell expresses CD86 and OX40L.In an embodiment, the invention includes a method of preparing any ofthe foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a CD86 protein comprising an amino acidsequence as set forth in SEQ ID NO:8 and a OX40L protein comprising anamino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of theMOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a CD86 protein comprisingan amino acid sequence as set forth in SEQ ID NO:8, and conservativeamino acid substitutions thereof, and a OX40L protein comprising anamino acid sequence as set forth in SEQ ID NO:10, and conservative aminoacid substitutions thereof, wherein the CD86 protein and the OX40Lprotein are expressed on the surface of the MOLM-13 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a CD86 protein comprising a sequence withgreater than 99% identity to an amino acid sequence as set forth in SEQID NO:8 and a OX40L protein comprising a sequence with greater than 99%identity to an amino acid sequence as set forth in SEQ ID NO:10, whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe MOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-14 cell modified to express a CD86 protein comprisinga sequence with greater than 98% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a OX40L protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:10, wherein the CD86 protein and the OX40L protein are expressedon the surface of the MOLM-14 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-14 cell modified to express a CD86protein comprising a sequence with greater than 97% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprisinga sequence with greater than 97% identity to an amino acid sequence asset forth in SEQ ID NO:10, wherein the CD86 protein and the OX40Lprotein are expressed on the surface of the MOLM-14 cell. In anembodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a CD86 protein comprising a sequence with greaterthan 96% identity to an amino acid sequence as set forth in SEQ ID NO:8and a OX40L protein comprising a sequence with greater than 96% identityto an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of theMOLM-14 cell. In an embodiment, the invention includes an aAPCcomprising a MOLM-14 cell modified to express a CD86 protein comprisinga sequence with greater than 95% identity to an amino acid sequence asset forth in SEQ ID NO:8 and a OX40L protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:10, wherein the CD86 protein and the OX40L protein are expressedon the surface of the MOLM-14 cell. In an embodiment, the inventionincludes an aAPC comprising a MOLM-14 cell modified to express a CD86protein comprising a sequence with greater than 90% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprisinga sequence with greater than 90% identity to an amino acid sequence asset forth in SEQ ID NO:10, wherein the CD86 protein and the OX40Lprotein are expressed on the surface of the MOLM-14 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In any of the foregoing embodiments, it will be understood that an aAPCcomprising a MOLM-14 or MOLM-13 cell may be modified to express bothOX40L and 4-1BBL.

The sequences for human CD86, human 4-1BBL (CD137L), and human OX40L(CD134L) are given in Table 3.

TABLE 3Amino acid sequences for human CD86, human 4-1BBL, and human OX40L.Identifier (Description) Sequence (One-Letter Amino Acid Symbols)SEQ ID NO: 8MGLSNILFVM AFLLSGAAPL KIQAYFNETA DLPCQFANSQ NQSLSELVVF WQDQENLVLN 60(human CD86)EVYLGKEKFD SVHSKYMGRT SFDSDSWTLR LHNLQIKDKG LYQCIIHHKK PTGMIRIHQM 120NSELSVLANF SQPEIVPISN ITENVYINLT CSSIHGYPEP KKMSVLLRTK NSTIEYDGIM 180QKSQDNVTEL YDVSISLSVS FPDVTSNMTI FCILETDKTR LLSSPFSIEL EDPQPPPDHI 240PWITAVLPTV IICVMVFCLI LWKWKKKKRP RNSYKCGTNT MEREESEQTK KREKIHIPER 300SDEAQRVFKS SKTSSCDKSD TCF 323 SEQ ID NO: 9MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA 60(human 4-1BBL,SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 120CD137) TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA180 LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV240 TPEIPAGLPS PRSE 254 SEQ ID NO: 10MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ 60(human OX40L,SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ 120CD134L)KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF 180CVL 183

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:13, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a first protein that bindsto a second protein comprising an amino acid sequence as set forth inSEQ ID NO:13, and conservative amino acid substitutions thereof, and athird protein that binds to a fourth protein comprising an amino acidsequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservativeamino acid substitutions thereof. In an embodiment, the inventionincludes a method of preparing any of the foregoing embodiments ofaAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13cell modified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:14, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes an aAPCcomprising a MOLM-13 cell modified to express a first protein that bindsto a second protein comprising an amino acid sequence as set forth inSEQ ID NO:14, and conservative amino acid substitutions thereof, and athird protein that binds to a fourth protein comprising an amino acidsequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservativeamino acid substitutions thereof. In an embodiment, the inventionincludes a method of preparing any of the foregoing embodiments ofaAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-14cell modified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-14 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising a MOLM-13cell modified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising a MOLM-13 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

The sequences for the ligands to which human CD86 binds (CD28 andCTLA-4), the ligand to which human 4-1BBL binds (4-1BB), and the ligandto which human OX40L binds (OX40) are given in Table 4.

TABLE 4Amino acid sequences for human CD28, human CTLA-4, human 4-1BB, and human OX40.Identifier (Description) Sequence (One-Letter Amino Acid Symbols)SEQ ID NO: 11MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 60(human CD28)SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 120PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 160SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS 220 SEQ ID NO: 12MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY 60(human CTLA-4)ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR 120AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL 180LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN 223 SEQ ID NO: 13MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 60(human 4-1BB)TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 120CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 180PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 240CSCRFPEEEE GGCEL 255 SEQ ID NO: 14MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 60(human OX40)NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 180GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 240RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 277

In an embodiment, the invention includes an isolated artificial antigenpresenting cell (aAPC) comprising a cell that expresses HLA-AB/C,ICOS-L, and CD58, and is modified to express one or more costimulatorymolecules, wherein the aAPC is derived from an EM-3 parental cell line.In an embodiment, the invention includes an aAPC comprising an EM-3 cellthat is modified to express one or more costimulatory molecules. In anembodiment, the invention includes an aAPC comprising an EM-2 cell thatis modified to express one or more costimulatory molecules.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellthat expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modifiedto express a CD86 protein comprising an amino acid sequence as set forthin SEQ ID NO:8, and conservative amino acid substitutions thereof, and a4-1BBL protein comprising an amino acid sequence as set forth in SEQ IDNO:9, and conservative amino acid substitutions thereof, and wherein theCD86 protein and the 4-1BBL protein are expressed on the surface of theEM-3 cell.

In an embodiment, the invention includes an aAPC comprising an EM-3 celltransduced with one or more viral vectors, wherein the one or more viralvectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the EM-3 cell expresses CD86 and 4-1BBL. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a CD86 protein comprising an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-3 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a CD86 protein comprising a sequence with greaterthan 99% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 99%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe EM-3 cell. In an embodiment, the invention includes an aAPCcomprising a EM-3 cell modified to express a CD86 protein comprising asequence with greater than 98% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the EM-3 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-3 cell modified to express a CD86protein comprising a sequence with greater than 97% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-3 cell. In anembodiment, the invention includes an aAPC comprising a EM-3 cellmodified to express a CD86 protein comprising a sequence with greaterthan 96% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 96%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe EM-3 cell. In an embodiment, the invention includes an aAPCcomprising a EM-3 cell modified to express a CD86 protein comprising asequence with greater than 95% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the EM-3 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-3 cell modified to express a CD86protein comprising a sequence with greater than 90% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-3 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:13, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes a methodof preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3modified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a single chain fragment variable (scFv) bindingdomain, such as clones 7C12 and 8B3 described herein, to bind the Fcdomain of a monoclonal antibody, such as OKT-3, providing an additionalproliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a CD86 protein comprising an amino acid sequence asset forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-2 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a CD86 protein comprising a sequence with greaterthan 99% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 99%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe EM-2 cell. In an embodiment, the invention includes an aAPCcomprising a EM-2 cell modified to express a CD86 protein comprising asequence with greater than 98% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the EM-2 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-2 cell modified to express a CD86protein comprising a sequence with greater than 97% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-2 cell. In anembodiment, the invention includes an aAPC comprising a EM-2 cellmodified to express a CD86 protein comprising a sequence with greaterthan 96% identity to an amino acid sequence as set forth in SEQ ID NO:8and a 4-1BBL protein comprising a sequence with greater than 96%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe EM-2 cell. In an embodiment, the invention includes an aAPCcomprising a EM-2 cell modified to express a CD86 protein comprising asequence with greater than 95% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the EM-2 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-2 cell modified to express a CD86protein comprising a sequence with greater than 90% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the EM-2 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:13, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes a methodof preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2modified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:13 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a single chain fragment variable (scFv) bindingdomain, such as clones 7C12 and 8B3 described herein, to bind the Fcdomain of a monoclonal antibody, such as OKT-3, providing an additionalproliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-3 oran EM-2 cell modified as depicted in FIG. 96. In an embodiment, theinvention includes an aAPC comprising an EM-3 or an EM-2 cell modifiedas depicted in FIG. 97. In an embodiment, the invention includes an aAPCcomprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellthat expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modifiedto express a CD86 protein comprising an amino acid sequence as set forthin SEQ ID NO:8, and conservative amino acid substitutions thereof, and aOX40L protein comprising an amino acid sequence as set forth in SEQ IDNO:10, and conservative amino acid substitutions thereof, and whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe EM-3 cell.

In an embodiment, the invention includes an aAPC comprising an EM-3 celltransduced with one or more viral vectors, wherein the one or more viralvectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding OX40L, and wherein the EM-3 cell expresses CD86 and OX40L. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a CD86 protein comprising an amino acid sequence asset forth in SEQ ID NO:8 and a OX40L protein comprising an amino acidsequence as set forth in SEQ ID NO:10, wherein the CD86 protein and theOX40L protein are expressed on the surface of the EM-3 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a CD86 protein comprising a sequence with greaterthan 99% identity to an amino acid sequence as set forth in SEQ ID NO:8and a OX40L protein comprising a sequence with greater than 99% identityto an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of the EM-3cell. In an embodiment, the invention includes an aAPC comprising a EM-3cell modified to express a CD86 protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:8 and a OX40L protein comprising a sequence with greater than 98%identity to an amino acid sequence as set forth in SEQ ID NO:10, whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe EM-3 cell. In an embodiment, the invention includes an aAPCcomprising a EM-3 cell modified to express a CD86 protein comprising asequence with greater than 97% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a OX40L protein comprising a sequence withgreater than 97% identity to an amino acid sequence as set forth in SEQID NO:10, wherein the CD86 protein and the OX40L protein are expressedon the surface of the EM-3 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-3 cell modified to express a CD86protein comprising a sequence with greater than 96% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprisinga sequence with greater than 96% identity to an amino acid sequence asset forth in SEQ ID NO:10, wherein the CD86 protein and the OX40Lprotein are expressed on the surface of the EM-3 cell. In an embodiment,the invention includes an aAPC comprising a EM-3 cell modified toexpress a CD86 protein comprising a sequence with greater than 95%identity to an amino acid sequence as set forth in SEQ ID NO:8 and aOX40L protein comprising a sequence with greater than 95% identity to anamino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of the EM-3cell. In an embodiment, the invention includes an aAPC comprising a EM-3cell modified to express a CD86 protein comprising a sequence withgreater than 90% identity to an amino acid sequence as set forth in SEQID NO:8 and a OX40L protein comprising a sequence with greater than 90%identity to an amino acid sequence as set forth in SEQ ID NO:10, whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe EM-3 cell. In an embodiment, the invention includes a method ofpreparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:14, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes a methodof preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3modified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-3 cellmodified to express a single chain fragment variable (scFv) bindingdomain, such as clones 7C12 and 8B3 described herein, to bind the Fcdomain of a monoclonal antibody, such as OKT-3, providing an additionalproliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a CD86 protein comprising an amino acid sequence asset forth in SEQ ID NO:8 and a OX40L protein comprising an amino acidsequence as set forth in SEQ ID NO:10, wherein the CD86 protein and theOX40L protein are expressed on the surface of the EM-2 cell. In anembodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a CD86 protein comprising a sequence with greaterthan 99% identity to an amino acid sequence as set forth in SEQ ID NO:8and a OX40L protein comprising a sequence with greater than 99% identityto an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of the EM-2cell. In an embodiment, the invention includes an aAPC comprising a EM-2cell modified to express a CD86 protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:8 and a OX40L protein comprising a sequence with greater than 98%identity to an amino acid sequence as set forth in SEQ ID NO:10, whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe EM-2 cell. In an embodiment, the invention includes an aAPCcomprising a EM-2 cell modified to express a CD86 protein comprising asequence with greater than 97% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a OX40L protein comprising a sequence withgreater than 97% identity to an amino acid sequence as set forth in SEQID NO:10, wherein the CD86 protein and the OX40L protein are expressedon the surface of the EM-2 cell. In an embodiment, the inventionincludes an aAPC comprising a EM-2 cell modified to express a CD86protein comprising a sequence with greater than 96% identity to an aminoacid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprisinga sequence with greater than 96% identity to an amino acid sequence asset forth in SEQ ID NO:10, wherein the CD86 protein and the OX40Lprotein are expressed on the surface of the EM-2 cell. In an embodiment,the invention includes an aAPC comprising a EM-2 cell modified toexpress a CD86 protein comprising a sequence with greater than 95%identity to an amino acid sequence as set forth in SEQ ID NO:8 and aOX40L protein comprising a sequence with greater than 95% identity to anamino acid sequence as set forth in SEQ ID NO:10, wherein the CD86protein and the OX40L protein are expressed on the surface of the EM-2cell. In an embodiment, the invention includes an aAPC comprising a EM-2cell modified to express a CD86 protein comprising a sequence withgreater than 90% identity to an amino acid sequence as set forth in SEQID NO:8 and a OX40L protein comprising a sequence with greater than 90%identity to an amino acid sequence as set forth in SEQ ID NO:10, whereinthe CD86 protein and the OX40L protein are expressed on the surface ofthe EM-2 cell. In an embodiment, the invention includes a method ofpreparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising an amino acid sequence as set forth in SEQ ID NO:14, andconservative amino acid substitutions thereof, and a third protein thatbinds to a fourth protein comprising an amino acid sequence as set forthin SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acidsubstitutions thereof. In an embodiment, the invention includes a methodof preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 99% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 98% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2modified to express a first protein that binds to a second proteincomprising a sequence with greater than 97% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 97% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 96% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 95% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a first protein that binds to a second proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:14 and a third protein that binds toa fourth protein comprising a sequence with greater than 90% identity toan amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. Inan embodiment, the invention includes a method of preparing any of theforegoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising an EM-2 cellmodified to express a single chain fragment variable (scFv) bindingdomain, such as clones 7C12 and 8B3 described herein, to bind the Fcdomain of a monoclonal antibody, such as OKT-3, providing an additionalproliferative signal.

In an embodiment, the invention includes an aAPC comprising an EM-3 oran EM-2 cell modified as depicted in FIG. 96. In an embodiment, theinvention includes an aAPC comprising an EM-3 or an EM-2 cell modifiedas depicted in FIG. 97. In an embodiment, the invention includes an aAPCcomprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.

In any of the foregoing embodiments, it is understood that an aAPCcomprising an EM-3 or EM-2 cell may be modified to express both OX40Land 4-1BBL.

In an embodiment, the invention includes an isolated artificial antigenpresenting cell (aAPC) comprising a cell that expresses CD58, and ismodified to express one or more costimulatory molecules, wherein theaAPC is derived from a K562-lineage parental cell line. In anembodiment, the invention includes an aAPC comprising a K562-lineagecell that is modified to express one or more costimulatory molecules. Inan embodiment, the K562 lineage parental cell line is deposited underaccession no. ATCC CCL-243 and also at European Collection ofAuthenticated. Cell Cultures (ECACCECACC 89121407).

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell that expresses CD58, wherein the cell is modified toexpress a CD86 protein comprising an amino acid sequence as set forth inSEQ ID NO:8, and conservative amino acid substitutions thereof, and a4-1BBL protein comprising an amino acid sequence as set forth in SEQ IDNO:9, and conservative amino acid substitutions thereof, and wherein theCD86 protein and the 4-1BBL protein are expressed on the surface of theK562-lineage cell.

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell transduced with one or more viral vectors, wherein theone or more viral vectors comprise a nucleic acid encoding CD86 and anucleic acid encoding 4-1BBL, and wherein the K562-lineage cellexpresses CD86 and 4-1BBL. In an embodiment, the invention includes amethod of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell modified to express a CD86 protein comprising an aminoacid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe K562-lineage cell. In an embodiment, the invention includes a methodof preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell modified to express a CD86 protein comprising asequence with greater than 99% identity to an amino acid sequence as setforth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence withgreater than 99% identity to an amino acid sequence as set forth in SEQID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressedon the surface of the K562-lineage cell. In an embodiment, the inventionincludes an aAPC comprising a K562-lineage cell modified to express aCD86 protein comprising a sequence with greater than 98% identity to anamino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL proteincomprising a sequence with greater than 98% identity to an amino acidsequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the4-1BBL protein are expressed on the surface of the K562-lineage cell. Inan embodiment, the invention includes an aAPC comprising a K562-lineagecell modified to express a CD86 protein comprising a sequence withgreater than 97% identity to an amino acid sequence as set forth in SEQID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97%identity to an amino acid sequence as set forth in SEQ ID NO:9, whereinthe CD86 protein and the 4-1BBL protein are expressed on the surface ofthe K562-lineage cell. In an embodiment, the invention includes an aAPCcomprising a K562-lineage cell modified to express a CD86 proteincomprising a sequence with greater than 96% identity to an amino acidsequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising asequence with greater than 96% identity to an amino acid sequence as setforth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL proteinare expressed on the surface of the K562-lineage cell. In an embodiment,the invention includes an aAPC comprising a K562-lineage cell modifiedto express a CD86 protein comprising a sequence with greater than 95%identity to an amino acid sequence as set forth in SEQ ID NO:8 and a4-1BBL protein comprising a sequence with greater than 95% identity toan amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86protein and the 4-1BBL protein are expressed on the surface of theK562-lineage cell. In an embodiment, the invention includes an aAPCcomprising a K562-lineage cell modified to express a CD86 proteincomprising a sequence with greater than 90% identity to an amino acidsequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising asequence with greater than 90% identity to an amino acid sequence as setforth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL proteinare expressed on the surface of the K562-lineage cell. In an embodiment,the invention includes a method of preparing any of the foregoingembodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell modified to express a first protein that binds to asecond protein comprising an amino acid sequence as set forth in SEQ IDNO:11, and conservative amino acid substitutions thereof, and a thirdprotein that binds to a fourth protein comprising an amino acid sequenceas set forth in SEQ ID NO:12 or SEQ ID NO:13, and conservative aminoacid substitutions thereof. In an embodiment, the invention includes amethod of preparing any of the foregoing embodiments of aAPCs.

In an embodiment, the invention includes an aAPC comprising aK562-lineage cell modified to express a first protein that binds to asecond protein comprising a sequence with greater than 99% identity toan amino acid sequence as set forth in SEQ ID NO:11 and a third proteinthat binds to a fourth protein comprising a sequence with greater than99% identity to an amino acid sequence as set forth in SEQ ID NO:12 orSEQ ID NO:13. In an embodiment, the invention includes an aAPCcomprising a K562-lineage cell modified to express a first protein thatbinds to a second protein comprising a sequence with greater than 98%identity to an amino acid sequence as set forth in SEQ ID NO:11 and athird protein that binds to a fourth protein comprising a sequence withgreater than 98% identity to an amino acid sequence as set forth in SEQID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes anaAPC comprising a K562-lineage modified to express a first protein thatbinds to a second protein comprising a sequence with greater than 97%identity to an amino acid sequence as set forth in SEQ ID NO:11 and athird protein that binds to a fourth protein comprising a sequence withgreater than 97% identity to an amino acid sequence as set forth in SEQID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes anaAPC comprising a K562-lineage cell modified to express a first proteinthat binds to a second protein comprising a sequence with greater than96% identity to an amino acid sequence as set forth in SEQ ID NO:11 anda third protein that binds to a fourth protein comprising a sequencewith greater than 96% identity to an amino acid sequence as set forth inSEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includesan aAPC comprising a K562-lineage cell modified to express a firstprotein that binds to a second protein comprising a sequence withgreater than 95% identity to an amino acid sequence as set forth in SEQID NO:11 and a third protein that binds to a fourth protein comprising asequence with greater than 95% identity to an amino acid sequence as setforth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the inventionincludes an aAPC comprising a K562-lineage cell modified to express afirst protein that binds to a second protein comprising a sequence withgreater than 90% identity to an amino acid sequence as set forth in SEQID NO:11 and a third protein that binds to a fourth protein comprising asequence with greater than 90% identity to an amino acid sequence as setforth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the inventionincludes a method of preparing any of the foregoing embodiments ofaAPCs.

In an embodiment, the invention includes an aAPC comprising anK562-lineage cell modified to express a single chain fragment variable(scFv) binding domain, such as clones 7C12 and 8B3 described herein, tobind the Fc domain of a monoclonal antibody, such as OKT-3, providing anadditional proliferative signal.

Methods of Preparing Artificial Antigen Presenting Cells

In an embodiment, a method of preparing an aAPC includes the step ofstable incorporation of genes for production of CD86 and 4-1BBL. In anembodiment, a method of preparing an aAPC includes the step ofretroviral transduction. In an embodiment, a method of preparing an aAPCincludes the step of lentiviral transduction. Lentiviral transductionsystems are known in the art and are described, e.g., in Levine, et al.,Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat.Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72,8463-71, and U.S. Pat. No. 6,627,442, the disclosures of each of whichare incorporated by reference herein. In an embodiment, a method ofpreparing an aAPC includes the step of gamma-retroviral transduction.Gamma-retroviral transduction systems are known in the art and aredescribed, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996,9.9.1-9.9.16, the disclosure of which is incorporated by referenceherein. In an embodiment, a method of preparing an aAPC includes thestep of transposon-mediated gene transfer. Transposon-mediated genetransfer systems are known in the art and include systems wherein thetransposase is provided as DNA expression vector or as an expressibleRNA or a protein such that long-term expression of the transposase doesnot occur in the transgenic cells, for example, a transposase providedas an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitabletransposon-mediated gene transfer systems, including the salmonid-typeTel-like transposase (SB or Sleeping Beauty transposase), such as SB10,SB11, and SB100×, and engineered enzymes with increased enzymaticactivity, are described in, e.g., Hackett, et al., Mol. Therapy 2010,18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of whichare incorporated by reference herein.

Examples of viruses modified and applied to such techniques includeadenoviruses, adeno-associated viruses, herpes simplex viruses, andretroviruses. Generally, nucleic acid molecules of interest may becloned into a viral genome. Upon replication and packaging of the viralgenome, the resultant viral particle is capable of delivering thenucleic acid of interest into a cell via the viral entry mechanism.

Of particular interest is the use of modified retroviruses to introducegenetic material into cells to treat genetic defects and other diseases.

The present invention provides highly efficient methods, andcompositions related thereto, for the stable transduction of cells withviral vectors and viral particles. By “stable transduction,” it is meantwhere an integrated form of the viral vector has been inserted into thechromosomal DNA of the transduced cell. The methods comprise exposingthe cells to be transduced to contact with at least one molecule thatbinds the cell surface. This contacting step may occur prior to, during,or after the cells are exposed to the viral vector or viral particle.Hereinafter, the term “viral vector” will be used to denote any form ofa nucleic acid derived from a virus and used to transfer geneticmaterial into a cell via transduction. The term encompasses viral vectornucleic acids, such as DNA and RNA, encapsidated forms of these nucleicacids, and viral particles in which the viral vector nucleic acids havebeen packaged.

Additional examples of cell surface binding molecules includepolypeptides, nucleic acids, carbohydrates, lipids, and ions, alloptionally complexed with other substances. Preferably, the moleculesbind factors found on the surfaces of blood cells, such as CD1a, CD1b,CD1c, CD1d, CD2, CD3γ, CD3δ, CD3∈, CD4, CD5, CD6, CD7, CD8α, CD8β, CD9,CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b,CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29,CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41,CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48,CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54,CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63,CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69,CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79α, CD79β, CD80,CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92,CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103,CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD114, CD115,CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122,CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b, CDw130, CDw131,CD 132, CD133, CD134, CD135, CD 136, CDw137, CD138, CD139, CD140a,CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149,CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b,CD161, CD162, CD163, CD164, CD165, CD166, and TCRξ. Small letters (e.g.“a” or “b”) indicate complex CD molecules composed of multiple geneproducts or belonging to families of structurally related proteins. Thenotation “w” refers to putative CD molecules that have not yet beenfully confirmed. A more complete listing of CD molecules is found inKishimoto, T. (ed). Current information on CD molecules is also found inShaw, S. (ed)., Protein Reviews on the Web: An International WWWResource/Journal at http://www.bsi.vt.edu/immunology.

More preferred are molecules that bind factors found on the Surfaces oflymphocytes, T cells and leukocytes, Such as CD2, CD3γ, CD3δ, CD3∈, CD5,CD6, CD7, CD8α, CD8β, CD9, CD11a, CD18, CD25, CD26, CD27, CD28, CD29,CD30, CD37, CD38, CD39, CD43, CD44, CD45R, CD46, CD48, CD49a, CD49b,CD49c, CD49d, CD49e, CD49f, CD50, CD53, CD54, CD56, CD57, CD58, CD59,CDw60, CD62L, CD68, CD69, CDw70, CD71, CD73, CDw75, CDw76, CD84, CD85,CD86, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD100, CD101,CD103, CD107a, CD107b, CDw108, CDw109, CD118, CD119, CD120b, CD121a,CD122, CDw124, CDw127, CDw128a, CDw130, CD132, CD134, CDw137, CD140a,CD140b, CD143, CD146, CD148, CD152, CD153, CD154, CD155, CD161, CD162,CD165, CD166, and TCRξ.

Of course any cell can be used in the practice of the invention.Preferably, the cell to be transduced is a eukaryotic cell. Morepreferably, the cell is a primary cell. Cell lines, however, may also betransduced with the methods of the invention and, in many cases, moreeasily transduced. In one preferred embodiment, the cell to betransduced is a primary lymphocyte (such as a T lymphocyte) or amacrophage (such as a monocytic macrophage), or is a precursor to eitherof these cells, such as a hematopoietic stem cell. Other preferred cellsfor transduction in general are cells of the hematopoietic system, or,more generally, cells formed by hematopoiesis as well as the stem cellsfrom which they form and cells associated with blood cell function. Suchcells include granulocytes and lymphocytes formed by hematopoiesis aswell as the progenitor pluripotent, lymphoid, and myeloid stem cells.Cells associated with blood cell function include cells that aid in thefunctioning of immune system cells, such as antigen presenting cellslike dendritic cells, endothelial cells, monocytes, and Langerhanscells. In a preferred embodiment, the cells are T lymphocytes (or Tcells), such as those expressing CD4 and CD8 markers.

In particularly preferred embodiments, the cell is a primary CD4+Tlymphocyte or a primary CD34+ hematopoietic stem cell. However, andgiven that the viral vectors for use in the invention may be pseudotypedwith Vesicular Stomatitis Virus envelope G protein (as discussed below),any cell can be transduced via the methods of the present invention.

Preferably, the cell is of a eukaryotic, multicellular species (e.g., asopposed to a unicellular yeast cell), and, even more preferably, is ofmammalian origin, e.g., a human cell.

Such a “larger collection of cells” can comprise, for instance, a cellculture (either mixed or pure).

Additional applications of the invention in cancer therapy are numerous,and one skilled in the art would be able to use the invention set outherein for the treatment of many types of cancers without undueexperimentation.

Furthermore, in Vivo uses are not restricted to disease states and canbe used to transduce normal cells. For example, the invention may beused to transduce hematopoietic stem cells in vivo in the bone marrow.Any combination of antibodies or other cell surface binding molecules,such as FLT-3 ligand, TPO and Kit ligand, or functional analogs thereof,or stromal cells expressing the cell surface binding molecule, could beadded with vector upon direct injection into the bone marrow for highefficiency bone marrow transduction.

Transduction of mainly a cell type of interest can be accomplished bythe choice of cell surface moiety to be bound. Thus in a mixedpopulation of blood cells, for example, transduction of cells expressingCD3, Such as certain T cells, will be enhanced when CD3 specific antibodies are used to interact with the cells. This will occur inpreference over other cell types in the population, such as granulocytesand monocytes that do not express CD3.

The invention also encompasses the transduction of purified or isolatedcell types if desired. The use of a purified or isolated cell typeprovides additional advantages Such as higher efficiencies oftransduction due to higher vector concentrations relative to the cell tobe transduced.

The present invention includes viral vectors, and compositionscomprising them, for use in the disclosed methods. The vectors arepreferably retroviral (family Retroviridae) vectors, and more preferablylentiviral vectors. Other retro viral vectors, such as oncoviral andmurine retroviral vectors, may also be used. Additional vectors may bederived from other DNA viruses or viruses that can convert their genomesinto DNA during some point of their lifecycle. Preferably the virusesare from the families Adenoviridae, Parvoviridae Hepandaviridae(including the hepatitis delta virus and the hepatitis E virus which isnot normally classified in the Hepandaviridae), Papoviridae (includingthe polyomavirinae and the papillomavirinae), Herpesviridae, andPoxviridae.

Additional viruses of the family Retroviridae (i.e., a retrovirus), areof the genus or subfamily Oncovirinae, Spumavirinae, Spumavirus,Lentivirinae, and Lentivirus. An RNA virus of the subfamily Oncovirinaeis desirably a human T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 orHTLV-2) or bovine leukemia virus (BLV), an avian leukosissarcoma virus(e.g., Rous Sarcoma virus (RSV), avian myeloblastosis virus (AMV), avianerythroblastosis virus (AEV), and Rous-associated virus (RAV; RAV-0 toRAV-50), a mammalian C-type virus (e.g., Moloney murine leukemia virus(Mul V), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemiavirus (A-MuLV), AKR-Mul V, feline leukemia virus (FeLV), simian sarcomavirus, reticuloendotheliosis virus (REV), Spleen necrosis virus (SNV)),a B-type virus (e.g., mouse mammary tumor virus (MMTV)), and a D-typevirus (e.g., Mason-Pfizer monkey virus (MPMV) and “SAIDS” viruses).

An RNA virus of the subfamily Lentivirus is desirably a humanimmunodeficiency virus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1was formerly called lymphadenopathy associated virus 3 (HTLV-III) andacquired immune deficiency syndrome (AIDS)-related virus (ARV)), oranother virus related to HIV-1 or HIV-2 that has been identified andassociated with AIDS or AIDS-like disease. The acronym “HIV” or terms“AIDS virus” or “human Immunodeficiency virus” are used herein to referto these HIV viruses, and HIV-related and -associated viruses,generically. Moreover, a RNA virus of the subfamily Lentiviruspreferably is a Visna/maedi virus (e.g., such as infect sheep), a felineimmunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiencyvirus (SIV), an equine infectious anemia virus (EIAV), and a caprinearthritisencephalitis virus (CAEV).

A particularly preferred lentiviral vector is one derived from HIV, mostpreferably HI-1, HIV-2, or chimeric combinations thereof. Of coursedifferent serotypes of retroviruses, especially HIV, may be used singlyor in any combination to prepare vectors for use in the presentinvention. Preferred vectors of the invention contains cis actingelements that are present in the wild-type virus, but not present in a“basic” lentiviral vector. A “basic” lentiviral vector containsminimally, LTRS and packaging sequences in the 5′ leader and gagencoding Sequences, but can also optionally contain the RRE element tofacilitate nuclear export of vector RNA in a Rev dependent manner. Apreferred vector additionally contains nucleotide aequences that enhancethe efficiency of transduction into cells.

An example of such a vector is pN2cGFP, a vector that contains thecomplete sequences of gag and pol. Another example is a vector thatcontain sequences from about position 4551 to position 5096 in pol(reference positions from the pNL4-3 sequence, Accession number M19921,HIVNL43 9709 bp, kindly provided by C. E. Buckler, NIAID, NIH, Bethesda,Md.). However any cis-acting sequence from the wt-HIV that can improvevector transduction efficiency may be used. Other examples of vectorscapable of efficient transduction via the present invention are cr2HIVconstructs as described in U.S. Pat. No. 5,885,806.

Additional examples of Viral vector constructs that may be used in thepresent invention are found in U.S. Pat. No. 5,885,806, which is herebyincorporated by reference as if fully set forth. The constructs in U.S.Pat. No. 5,885,806 are merely examples that do not limit the scope ofvectors that efficiently transduce cells. Instead, the constructsprovide additional guidance to the skilled artisan that a viral vectorfor use with the present invention may contain minimal sequences fromthe wild-type virus or contain sequences up to almost the entire genomeof wild-type virus, yet exclude an essential nucleic acid sequencerequired for replication and/or production of disease. Methods fordetermining precisely the sequences required for efficient transductionof cells are routine and well known in the art. For example, asystematic incorporation of viral sequences back into a “basic” vectoror deleting sequences from vectors that contain virtually the entire HIVgenome, such as cr2HIVs, is routine and well known in the art.

Furthermore, placing sequences from other viral back bones into viralvectors of interest, such as the cytomegalovirus (CMV), is also wellknown in the art. Regardless of the actual viral vector used, variousaccessory proteins encoded by, and sequences present in, the viralgenetic material may be left in the vector or helper genomes if theseproteins or sequences increase transduction efficiency in certain celltypes. Numerous routine screens are available to determine whethercertain genetic material increases transduction efficiency byincorporating the sequence in either the vector or helper genomes. Apreferred embodiment of the invention is to not include accessoryproteins in either the vector or helper genomes. But this preferencedoes not exclude embodiments of the invention where accessory proteinsand other sequences are left in either the vector or a helper genome toincrease transduction efficiency.

The viral vector for use in the transduction methods of the inventioncan also comprise and express one or more nucleic acid sequences underthe control of a promoter present in the virus or under the control of aheterologous promoter introduced into the vector. The promoters mayfurther contain insulatory elements, such as erythroid DNAsehyper-sensitive sites, so as to flank the operon for tightly controlledgene expression. Preferred promoters include the HIV-LTR, CMV promoter,PGK, Ul, EBER transcriptional units from Epstein Barr Virus, tRNA, U6and U7. While Pol II promoters are preferred, Pol III promoters may alsobe used. Tissue specific promoters are also preferred embodiments. Forexample, the beta globin Locus Control Region enhancer and the alpha &beta globin promoters can provide tissue specific expression inerythrocytes and erythroid cells. Another further preferred embodimentis to use cis-acting sequences that are associated with the promoters.For example, The Ul gene may be used to enhance antisense geneexpression where non-promoter sequences are used to target the antisenseor ribozymes molecule to a target spliced RNA as set out in U.S. Pat.No. 5,814,500, which is hereby incorporated by reference.

Such sequences and gene products are preferably biologically activeagents capable of producing a biological effect in a cell.

In one preferred embodiment, the agent is a cell surface molecule.

In the methods of the invention, the cells to be transduced are exposedto contact with the at least one molecule that binds the cell surfacebefore, after, or simultaneously with application of the viral vector.For example, the cells can be cultured in media with CD3 and CD28antibodies (coated onto the surface of the culture dish or immobilizedon beads present in the culture) before, after, or in the presence ofthe viral vector to be transduced. Preferably, the cells are exposed toimmobilized CD3 and/or CD28 only after or only upon initial contact withthe viral vector. Under these conditions, the cells are not exposed tocell surface binding molecule(s) prior to actual transduction with theviral vector. In embodiments where contact with a cell surface bindingmolecule occurs after exposure of the cells to a viral vector(transduction), the contact preferably occurs within three days oftransduction, more preferably within one to two days after transduction.

Incubation of the cells with the viral vector may be for differentlengths of time, depending on the conditions and materials used. Factorsthat influence the incubation time include the cell, vector and MOI(multiplicity of infection) used, the molecule(s) and amounts used tobind the cell surface, whether and how said molecule(s) are immobilizedor solubilized, and the level of transduction efficiency desired.Preferably, the incubation is for about eight to about 72 hours, morepreferably for about 12 to about 48 hours. In a particularly preferredembodiment, the incubation is for about 24 hours and is optionallyrepeated once.

Contact between the cells to be transduced and a viral vector occurs atleast once, but it may occur more than once, depending upon the celltype. For example, high efficiency transduction of CD34 positive stemcells have been accomplished with multiple transductions with vector. Apreferred method of the invention is to Simultaneously introduce a viralvector in combination with a cell surface binding molecule (e.g. CD3and/or CD28 antibodies or a FLT-3 ligand, TPO or Kit ligand) and avoidchanging the medium for between about one and about eight days aftertransduction. More preferably, the medium is not changed for three dayspost transduction. Transduction can proceed for as long as theconditions permit without the process being significantly detrimental tothe cells or the organism containing them. Additional examples of cellsurface binding proteins for such use include those describedhereinabove.

Similarly, the MOI used is from about 1 to about 400, preferably lessthan 500. Generally, the preferred MOI is from about 2 to about 50. Morepreferably, the MOI is from about 10 to about 30, although ranges offrom about 1 to about 10, about 20, about 30, or about 40 are alsocontemplated. Most preferred is an MOI of about 20. Furthermore, thecopy number of viral vector per cell should be at least one. However,many copies of the vector per cell may also be used with the abovedescribed methods. The preferred range of copies per cell is from about1 to about 100. The more preferred copy number is the minimum copynumber that provides a therapeutic, prophylactic or biological impactresulting from vector transduction or the most efficient transduction.

For therapeutic or prophylactic applications, a more preferred copynumber is the maximum copy number that is tolerated by the cell withoutbeing significantly detrimental to the cell or the organism containingit. Both the minimum and maximum copy number per cell will varydepending upon the cell to be transduced as well as other cells that maybe present. The optimum copy number is readily determined by thoseskilled in the art using routine methods. For example, cells aretransduced at increasing increments of concentration or multiplicitiesof infection. The cells are then analyzed for copy number, therapeuticor biological impact and for detrimental effects on the transduced cellsor a host containing them (e.g. safety and toxicity).

After incubation with the viral vector in vitro, the cells may becultured in the presence of the cell surface binding molecule(s) forvarious times before the cells are analyzed for the efficiency oftransduction or otherwise used. Alternatively, the cells may be culturedunder any conditions that result in cell growth and proliferation, Suchas incubation with interleukin-2 (IL-2) or incubation with the cellsurface binding molecule(s) followed by IL-2.

The efficiency of transduction observed with the present invention isfrom about 75-100%. Preferably, the efficiency is at least about 75 to90%. More preferred embodiments of the invention are where transductionefficiency is at least about 90 to 100%. Most preferred embodiments havetransduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97, 98, 99and 100%. In addition to the above, the transduced cells may be used inresearch or for treatment or prevention of disease conditions in livingsubjects.

Therapeutic uses for the transduced cells include the introduction ofthe cells into a living organism. For example, unstimulated primary Tcells isolated from an individual infected with, or at risk of beinginfected with HIV, may be first transduced by a vector, like thatdescribed in U.S. Pat. No. 5,885,806, using the present methods andfollowed by injection of the transduced cells back into the individual.

The present invention is directed to methods, and compositions relatedthereto, for the stable transduction of cells with viral vectors toefficiencies of greater than about 75%. Stably transduced cells may bedistinguished from transiently transduced, or pseudotransduced cells,after about seven to ten days, or optionally after about 14 days, posttransduction. The methods relate to the fact that contact of the cellsto be transduced with at least one molecule that binds the cell surfaceincreases the efficiency of stable transduction.

The methods of the invention comprise the step of transduction with aviral vector in combination with contact with a cell surface bindingmolecule. As noted above, the contact may occur before, after or at thesame time as transduction with the vector. The invention is broadlyapplicable to any cell, and the use of any cell surface bindingmolecule. Cells for use with the present methods include unstimulatedprimary cells, which are freshly isolated from an in vivo source as wellas cell lines, which may have been previously cultured for various timesin the presence of factors which maintain them in a proliferating state.

In the case of primary cells, they are first obtained from an in vivosource followed optionally by selection for particular cell types. Forexample, if primary CD4+ and/or CD8+ T cells are to be used, peripheralblood (PB) or cord blood (“CB” from an umbilical source) samples arefirst obtained followed by enrichment for CD4+ and/or CD8+ cell types.Standard magnetic beads positive selection, plastic adherence negativeselection, and/or other art recognized standard techniques may be usedto isolate CD4+ and/or CD8+ cells away from contaminating PB cells.Purity of the isolated cell types may be determined by immunophenotypingand flow cytometry using standard techniques.

After isolation, the primary cells may be used in the present methods tobe transduced with Viral vectors at efficiencies of greater than 75%.The invention is most advantageously used with primary lymphocytes, Suchas T cells, transduced with an HIV-1 based vector capable of expressingheterologous genetic material of interest. Another preferred use is withprimary hematopoietic stem cells, such as CD34 positive cells. In caseswhere the heterologous genetic material is or encodes a therapeutic orprophylactic product for use in vivo to treat or prevent a disease, thetransduced primary cell can be introduced back into an in vivoenvironment, such as a patient. As such, the invention contemplates theuse of the transfected cells in gene therapy to treat, or prevent, adisease by combating a genetic defect or targeting a viral infection.

For the transduction of primary cells in a mixed population, the aboveisolation/purification steps would not be used. Instead, the cell to betransduced would be targeted by selection of at least one appropriatecell surface molecule or moiety found on that cell type and thepreparation of one or more molecules capable of binding said moiety. Thecell surface moiety may be a receptor, marker, or other recognizableepitope on the surface of the targeted cells. Once selected, moleculesthat interact with the moiety, such as specific antibodies, may beprepared for use in the present invention.

For example, CD4+ and/or CD8+ cells can either be first purified andthen transduced by the methods of the invention with the use ofimmobilized CD3 and CD28 antibodies or alternatively be transduced aspart of a mixed population, like peripheral blood cells (PBCs) orperipheral blood mononuclear cells (PBMNCs), by use of the sameantibodies. Hematopoietic stem cells in total white blood cellpopulations, which may be difficult to purify or isolate, may betransduced in the mixed populations by use of immobilized CD34antibodies.

The cell surface binding molecules of the invention may target and bindany moiety found on the surface of the cell to be transduced.Preferably, the moieties are found as part of receptors, markers, orother proteinaceous or nonproteinaceous factors on the cell Surface. Themoieties include epitopes recognized by the cell surface bindingmolecule. These epitopes include those comprising a polypeptidesequence, a carbohydrate, a lipid, a nucleic acid, an ion andcombinations thereof.

Examples of cell surface binding molecules include an antibody or anantigen binding fragment thereof and a ligand or binding domain for acell surface receptor. The cell surface binding molecule may itself be apolypeptide, a nucleic acid, a carbohydrate, a lipid, or an ion.Preferably, the molecule is an antibody or a fragment thereof, such as aFab, or F, fragment. More preferably, the molecule is not used in asoluble form but is rather immobilized on a solid medium, such a bead,with which the cells to be transduced may be cultured, or the surface ofa tissue culture dish, bag or plate, upon which the cells to betransduced may be cultured. In a preferred embodiment for thetransduction of CD4+ or CD8+ cells, monoclonal antibodies that recognizeCD3 and/or CD28 may be used in a cell culture bag in the presence of aviral vector.

The present invention includes compositions comprising a cell surfacebinding molecule for use as part of the disclosed methods. An exemplarycomposition comprises the molecule and a viral vector to be transduced,optionally in the presence of the cells to be transduced. The viralvectors may be derived from any source, but are preferably retroviralvectors. More preferably, they are lentiviral vectors. A particularlypreferred lentiviral vector is one derived from a Human ImmunodeficiencyVirus (HIV), most preferably HIV-1, HIV-2, or chimeric combinationsthereof. Of course different viral vectors may be simultaneouslytransduced into the same cell by use of the present methods. Forexample, one vector can be a replication deficient or conditionallyreplicating retroviral vector while a second vector can be a packagingconstruct that permits the first vector to be replicated/packaged andpropagated. When various viral accessory proteins are to be encoded by aviral vector, they may be present in any one of the vectors beingtransduced into the cell. Alternatively, the viral accessory proteinsmay be present in the transduction process via their presence in theviral particles used for transduction. Such viral particles may have aneffective amount of the accessory proteins co-packaged to result in anincrease in transduction efficiency. In a preferred embodiment, theviral vector does not encode one or more of the accessory proteins.

A viral vector for use in the transduction methods of the invention canalso comprise and express one or more nucleic acid sequences under thecontrol of a promoter. In one embodiment of the invention, a nucleicacid sequence encodes a gene product that, upon expression, wouldalleviate or correct a genetic deficiency in the cell to be transduced.In another embodiment, the nucleic acid sequence encodes or constitutesa genetic antiviral agent that can prevent or treat viral infection. By“genetic antiviral agent”, it is meant any substance that is encoded orconstituted by genetic material. Examples of such agents are provided inU.S. Pat. No. 5,885,806. They include agents that function by inhibitingviral proteins, such as reverse transcriptase or proteases, competingwith viral factors for binding or target sites, or targeting viraltargets directly for degradation, Such as in the case of ribozymes andantisense constructs. Other examples of genetic antiviral agents includeantisense, RNA decoys, transdominant mutants, interferons, toxins,nucleic acids that modulate or modify RNA splicing, immunogens, andribozymes, such as “hammerhead” and external guide sequence (EGS)mediated forms thereof.

The cells to be transduced may be exposed to contact with the viralvector either before, after or simultaneously with contact with the cellsurface binding molecule. Thus the cells can be first exposed to thevector for a period of time followed by introduction of the cell surfacebinding molecule. Such cells may be newly isolated or prepared primarycells that have not been intentionally stimulated to enter the cellcycle. Alternatively, the cells can be first exposed to the cell surfacebinding molecule for a period of time followed by contact with the viralvector. After contact with the vector, excess vector is preferably notremoved and the cells cultured under conditions conducive to cell growthand/or proliferation. Such conditions may be in the presence of the cellsurface binding molecule or other stimulatory/activating factors, suchas cytokines and lymphokines in the case of T cells. Alternatively,excess vector may be removed after contact with the cell and beforefurther culturing.

Another embodiment of the invention is to culture the cells in thepresence of both viral vector and cell surface binding moleculesimultaneously. Such cells are preferably not previously stimulated.After a period of time, the cells are cultured under growth orproliferation inducing conditions such as the continued presence of thecell surface binding molecule or other stimulatory/activating factors.Alternatively, excess vector may be removed before further culturing.

Incubation of the cells to be transduced with the viral vector may befor different lengths of time, depending on the conditions and materialsused. Factors that influence the incubation time include the cell,vector and MOI (multiplicity of infection) used, the molecule(s) andamounts used to bind the cell surface, whether and how said molecule(s)are immobilized, and the level of transduction efficiency desired. In apreferred embodiment of the invention, the cells are T lymphocytes, thevector HIV based, the MOI is about 20, the cell Surface bindingmolecules are CD3 and CD28 antibodies immobilized on beads, and theresultant efficiency at least 93%. As would be evident to the skilledperson in the art, some of the above factors are directly correlatedwhile others are inversely correlated. For example, a decrease in theMOI will likely decrease the level of efficiency while efficiency canlikely be maintained if an increased amount of cell surface bindingmolecules is used.

The length of incubation viral vector and the cells to be transformed ispreferably for 24 hours and optionally repeated once for lymphocytes andup to four times for hematopoietic stem cells. Similarly, and inembodiments where the cells are incubated with the cell surface bindingmolecule before introduction of the viral vector, the incubation may befor about 12 hours to about 96 hours. Preferably, incubation with a cellsurface binding molecule occurs simultaneously with contact of the cellswith the viral vector. Under such circumstances, the cell surfacebinding molecules may be left in contact with the cells when the vectoris introduced. Alternatively, excess cell surface binding molecules maybe first removed from the culture before introduction of the vector tothe cells.

After contact with the vector, the cells are cultured under conditionsconducive to their growth or proliferation. Preferably, the conditionsare continued culturing in the presence of the cell surface bindingmolecules. Alternatively, the cells are initially cultured with the cellsurface binding molecule followed by substitution with media containinganother factor conducive to cell growth, such as interleukin-2. Yetanother embodiment would be to remove both the excess cell surfacebinding molecule and the excess vector followed by culturing in thepresence of a factor conducive to growth or proliferation as well asenhancing further vector transduction. Such factors include mitogenssuch as phytohemaglutinin (PHA) and cytokines, growth factors,activators, cell surface receptors, cell surface molecules, solublefactors, or combinations thereof, as well as active fragments of suchmolecules, alone or in combination with another protein or factor, orcombinations thereof.

Examples of additional factors include epidermal growth factor (EGF),transforming growth factor alpha (TGF-alpha), angiotensin, transforminggrowth factor beta (TGF-beta), GDF, bone morphogenic protein (BMP),fibroblast growth factor (FGF acidic and basic), vascular endothelialgrowth factor (VEGF), PIGF, human growth hormone (HGH), bovine growthhormone (BGH), heregulins, amphiregulin, Ach receptor inducing activity(ARIA), RANTES (regulated on activation, normal T expressed andsecreted), angiogenins, hepatocyte growth factor, tumor necrosis factorbeta (TNF-beta), tumor necrosis factor alpha (TNF-alpha), angiopoietins1 or 2, insulin, insulin growth factors I or II (IGF-I or IGF-2),ephrins, leptins, interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or 15 (IL-1, IL-2, IL-3, IL-4, L-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, or IL-15), G-CSF (granulocyte colonystimulating factor), GM-CSF (granulocyte-macrophage colony stimulatingfactor), M-CSF (macrophage colony stimulating factor), LIF (leukemiainhibitory factor), angiostatin, oncostatin, erythropoietin (EPO),interferon alpha (including subtypes), interferons beta, gamma, andomega, chemokines, macrophage inflammatory protein-I alpha or beta(MIP-1 alpha or beta), monocyte chemotactic protein-1 or -2 (MCP-1 or2), GRO beta, MWF (macrophage migration inhibitory factor), MGSA(melanoma growth stimulatory activity), alpha inhibin HGF, PD-ECGF,bFGF, lymphotoxin, Mullerian inhibiting substance, FAS ligand,osteogenic protein, pleiotrophin/midkine, ciliary neurotrophic factor,androgen induced growth factor, autocrine motility factor, hedgehogprotein, estrogen, progesterone, androgen, glucocorticoid receptor,RAR/RXR, thyroid receptor, TRAP/CD40, EDF (erythroid differentiatingfactor), Fic (growth factor inducible chemokine), IL-1RA, SDF, NGR orRGD ligand, NGF, thymosine-alphal, OSM, chemokine receptors, Stem cellfactor (SCF), or combinations thereof. As evident to one skilled in theart, the choice of culture conditions will depend on knowledge in theart concerning the cells transduced as well as the subsequent intendeduse of the cells. For example, the combination of IL-3, IL-6 and stemcell factor would not be a choice for transduced cells that are to beused in human transplantation. Similarly, the choice of cultureconditions would preferably not be to the detriment of cell viability ortransduction efficiency.

Preferably, the post transduction incubation is for a period of aboutfour hours, or for about one to about seven to ten days. More preferablyfrom about 16 to about 20 hours or for about four, about five or aboutsix days. About fourteen days of post-transduction incubation is alsocontemplated.

The efficiency of transduction observed with the present invention isfrom about 75-100%. Preferably, the efficiency is at least about 75 to90%. More preferred embodiments of the invention are where transductionefficiency is at least about 90 to 95%. The most preferred embodimentshave transduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97,98, 99 and 100%.

In addition to the above, the transduced cells may be used in researchor for treatment of disease conditions in living subjects. Particularlypreferred as part of the invention are therapeutic uses for thetransduced cells to produce gene products of interest or for directintroduction into a living organism as part of gene therapy. Forexample, and as exemplified below, primary T cells can be isolated andtransduced with a viral vector.

In another embodiment, the T cells are transduced with genes or nucleicacids capable of conditionally killing the T cell upon introduction intoa host organism. This has applications in allogenic bone marrowtransplantation to prevent graft versus host disease by killing T cellswith a pro-drug approach.

Alternatively, the primary cells can be deficient in a gene product, andthe deficiency correctable by the transduced viral vector. Such cellswould be reintroduced into the living subject after transduction withthe vector.

Thus, both in vitro and ex vivo applications of the invention arecontemplated. For transfers into a living subject, the transduced cellsare preferably in a biologically acceptable solution or pharmaceuticallyacceptable formulation. Such a transfer may be made intravenously,intraperitoneally or by other injection and non-injection methods knownin the art. The dosages to be administered will vary depending on avariety of factors, but may be readily determined by the skilledpractitioner. There are numerous applications of the present invention,with known or well designed payloads in the viral vector, where thebenefits conferred by the transduced genetic material will outweigh anyrisk of negative effects.

In an embodiment, a method of preparing an aAPC includes the step ofstable incorporation of genes for transient production of CD86 and4-1BBL. In an embodiment, a method of preparing an aAPC includes thestep of electroporation. Electroporation methods are known in the artand are described, e.g., in Tsong, Biophys. 1 1991, 60, 297-306, andU.S. Patent Application Publication No. 2014/0227237 A1, the disclosuresof each of which are incorporated by reference herein. In an embodiment,a method of preparing an aAPC includes the step of calcium phosphatetransfection. Calcium phosphate transfection methods (calcium phosphateDNA precipitation, cell surface coating, and endocytosis) are known inthe art and are described in Graham and van der Eb, Virology 1973, 52,456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; andChen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Pat.No. 5,593,875, the disclosures of each of which are incorporated byreference herein. In an embodiment, a method of preparing an aAPCincludes the step of liposomal transfection. Liposomal transfectionmethods, such as methods that employ a 1:1 (w/w) liposome formulation ofthe cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filteredwater, are known in the art and are described in Rose, et al.,Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad.Sci. USA, 1987, 84, 7413-7417 and in U.S. Pat. Nos. 5,279,833;5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, thedisclosures of each of which are incorporated by reference herein. In anembodiment, a method of preparing an aAPC includes the step oftransfection using methods described in U.S. Pat. Nos. 5,766,902;6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of eachof which are incorporated by reference herein.

In an embodiment, the aAPC is transduced by first using the Gatewaycloning method (commercially available from ThermoFisher, Inc.) toprepare vector for lentiviral transduction, followed by lentiviraltransduction using the vector and one or more associated helperplasmids, as is also described elsewhere herein. In the Gateway cloningmethod, a gene is selected (such as CD86) and is then provided withprimers and amplified using PCR technology with the help of an attBtagged primer pair. The PCR fragment is then combined with a donorvector (pDONR, such as pDONR221) that includes attP sites to provide anentry clone, using the BP reaction. An integration reaction between theattB and the attP sites combines the PCR fragment with the donor vector.The resulting entry clone contains the gene of interest flanked by attLsites. The LR reaction is then used to combine the entry clone with adestination vector to produce an expression vector. In the LR reaction,a recombination reaction is used to link the entry clone with thedestination vector (such as pLV430G) using the attL and attR sites and aclonase enzyme. The attL sites are already found in the entry clone,while the destination vector includes the attR sites. The LR reaction iscarried out to transfer the sequence of interest into one or moredestination vectors in simultaneous reactions.

In some embodiments, the aAPCs described herein may be grown andmaintained under serum-based media and/or serum free media. According toan exemplary method, aAPCs may be cultured in 24 well plates at a celldensity of about 1×10⁶ cells per well for 3 to 5 days. The cells maythen be isolated and/or washed by centrifugation and resuspended inmedia or cryopreserved in an appropriate cryopreservation media (e.g.,CryoStor 10 (BioLife Solutions)) and stored in a −80° C. freezer.

In some embodiments, the aAPCs described herein may be grown in thepresence of serum-based media. In some embodiments, the aAPCs describedherein by may be grown in the presence of serum-based media thatincludes human serum (hSerum) containing media (e.g., cDMEM with 10%hSerum). In some embodiments, the aAPCs grown in the presence ofserum-based media may be selected from the group consisting of aMOLM-13cells, aMOLM-14 cells, and aEM3 cells.

In some embodiments, the aAPCs described herein may be grown in thepresence of serum free media. In some embodiments, the serum free mediamay be selected from the group consisting of CTS Optmizer(ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), XFSM(MesenCult), and the like. In some embodiments, the aAPCs grown in thepresence of serum free media may be selected from the group consistingof aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.

Methods of Expanding Tumor Infiltrating Lymphocytes and T Cells

In an embodiment, the invention includes a method of expanding tumorinfiltrating lymphocytes (TILs), the method comprising contacting apopulation of TILs comprising at least one TIL with an aAPC describedherein, wherein said aAPC comprises at least one co-stimulatory ligandthat specifically binds with a co-stimulatory molecule expressed on thecellular surface of the TILs, wherein binding of said co-stimulatorymolecule with said co-stimulatory ligand induces proliferation of theTILs, thereby specifically expanding TILs.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs) using any of theaAPCs of the present disclosure, the method comprising the steps asdescribed in Jin, et al., J. Immunotherapy 2012, 35, 283-292, thedisclosure of which is incorporated by reference herein. For example,the tumor may be placed in enzyme media and mechanically dissociated forapproximately 1 minute. The mixture may then be incubated for 30 minutesat 37° C. in 5% CO₂ and then mechanically disrupted again forapproximately 1 minute. After incubation for 30 minutes at 37° C. in 5%CO₂, the tumor may be mechanically disrupted a third time forapproximately 1 minute. If after the third mechanical disruption, largepieces of tissue are present, 1 or 2 additional mechanical dissociationsmay be applied to the sample, with or without 30 additional minutes ofincubation at 37° C. in 5% CO₂. At the end of the final incubation, ifthe cell suspension contains a large number of red blood cells or deadcells, a density gradient separation using Ficoll may be performed toremove these cells. TIL cultures were initiated in 24-well plates(Costar 24-well cell culture cluster, flat bottom; Corning Incorporated,Corning, N.Y.), each well may be seeded with 1×10⁶tumor digest cells orone tumor fragment approximately 1 to 8 mm³ in size in 2 mL of completemedium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). CMconsists of RPMI 1640 with GlutaMAX, supplemented with 10% human ABserum, 25 mM Hepes, and 10 mg/mL gentamicin. Cultures may be initiatedin gas-permeable flasks with a 40 mL capacity and a 10 cm² gas-permeablesilicon bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, eachflask may be loaded with 10-40×10⁶ viable tumor digest cells or 5-30tumor fragments in 10-40 mL of CM with IL-2. G-Rex 10 and 24-well platesmay be incubated in a humidified incubator at 37° C. in 5% CO₂ and 5days after culture initiation, half the media may be removed andreplaced with fresh CM and IL-2 and after day 5, half the media may bechanged every 2-3 days. Rapid expansion protocol (REP) of TILs may beperformed using T-175 flasks and gas-permeable bags or gas-permeableG-Rex flasks, as described elsewhere herein, using the aAPCs of thepresent disclosure. For REP in T-175 flasks, 1×10⁶ TILs may be suspendedin 150 mL of media in each flask. The TIL may be cultured with aAPCs ofthe present disclosure at a ratio described herein, in a 1 to 1 mixtureof CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL ofIL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The T-175 flasks may beincubated at 37° C. in 5% CO₂. Half the media may be changed on day 5using 50/50 medium with 3000 IU/mL of IL-2. On day 7, cells from 2 T-175flasks may be combined in a 3L bag and 300 mL of AIM-V with 5% human ABserum and 3000 IU/mL of IL-2 may be added to the 300 mL of TILsuspension. The number of cells in each bag may be counted every day ortwo days, and fresh media may be added to keep the cell count between0.5 and 2.0×10⁶ cells/mL. For REP in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (e.g., G-Rex 100, Wilson WolfManufacturing, as described elsewhere herein), 5×10⁶ or 10×10⁶ TILs maybe cultured with aAPCs at a ratio described herein (e.g., 1 to 100) in400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30ng/mL of anti-CD3 antibody (OKT-3). The G-Rex100 flasks may be incubatedat 37° C. in 5% CO₂. On day five, 250 mL of supernatant may be removedand placed into centrifuge bottles and centrifuged at 1500 rpm (491 g)for 10 minutes. The obtained TIL pellets may be resuspended with 150 mLof fresh 50/50 medium with 3000 IU/mL of IL-2 and added back to theG-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, onday seven the TIL in each G-Rex100 are suspended in the 300 mL of mediapresent in each flask and the cell suspension may be divided into three100 mL aliquots that may be used to seed 3 G-Rex100 flasks. About 150 mLof AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may then be addedto each flask. G-Rex100 flasks may then be incubated at 37° C. in 5%CO₂, and after four days, 150 mL of AIM-V with 3000 IU/mL of IL-2 may beadded to each G-Rex100 flask. After this, the REP may be completed byharvesting cells on day 14 of culture.

As described herein, TILs may be expanded advantageously in the presenceof serum free media. In some embodiments, the TIL expansion methodsdescribed herein may include the use of serum free media rather thanserum-based media (e.g., complete media or CM1). In some embodiments,the TIL expansion methods described herein may use serum free mediarather than serum-based media. In some embodiments, the serum free mediamay be selected from the group consisting of CTS Optmizer(ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), and thelike.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the cell culture medium further comprises IL-2 at an        initial concentration of about 3000 IU/mL and OKT-3 antibody at        an initial concentration of about 30 ng/mL.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the population of APCs expands the population of TILs by        at least 50-fold over a period of 7 days in a cell culture        medium.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the myeloid cell endogenously expresses HLA-AB/C,        ICOS-L, and CD58.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the myeloid cell is a MOLM-14 cell.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the myeloid cell is a MOLM-13 cell.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (c) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (d) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the myeloid cell is a EM-3 cell.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the CD86 protein comprises an amino acid sequence as set        forth in SEQ ID NO:8, or conservative amino acid substitutions        thereof, and the 4-1BBL protein comprises an amino acid sequence        as set forth in SEQ ID NO:9, or conservative amino acid        substitutions thereof.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the nucleic acid encoding CD86 comprises a nucleic acid        sequence as set forth in SEQ ID NO:19 and the nucleic acid        encoding 4-1BBL comprises a nucleic acid sequence as set forth        in SEQ ID NO:16.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the expansion is performed using a gas permeable        container.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the ratio of the population of TILs to the population of        aAPCs is between 1 to 200 and 1 to 400.

In an embodiment, the invention provides a method of expanding apopulation of tumor infiltrating lymphocytes (TILs), the methodcomprising the steps of:

-   -   (a) transducing a myeloid cell with one or more viral vectors to        obtain a population of artificial antigen presenting cells        (aAPCs), wherein the one or more viral vectors comprise a        nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL,        and wherein the myeloid cell expresses a CD86 protein and a        4-1BBL protein, and    -   (b) contacting the population of TILs with the population of        aAPCs in a cell culture medium,    -   wherein the ratio of the population of TILs to the population of        aAPCs is about 1 to 300.

In an embodiment, the invention provides a method of expanding tumorinfiltrating lymphocytes (TILs), the method comprising contacting apopulation of TILs comprising a population of TILs with a myeloidartificial antigen presenting cell (aAPC), wherein the myeloid aAPCcomprises at least two co-stimulatory ligands that specifically bindwith at least two co-stimulatory molecule on the TILs, wherein bindingof the co-stimulatory molecules with the co-stimulatory ligand inducesproliferation of the TILs, thereby specifically expanding TILs, andwherein the at least two co-stimulatory ligands comprise CD86 and4-1BBL.

In any of the foregoing embodiments, the aAPC may further comprise OX40Lin addition to 4-1BBL, or may comprise OX40L instead of 4-1BBL.

In an embodiment, a method of expanding or treating a cancer includes astep wherein TILs are obtained from a patient tumor sample. A patienttumor sample may be obtained using methods known in the art. Forexample, TILs may be cultured from enzymatic tumor digests and tumorfragments (about 1 to about 8 mm³ in size) from sharp dissection. Suchtumor digests may be produced by incubation in enzymatic media (e.g.,Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase)followed by mechanical dissociation (e.g., using a tissue dissociator).Tumor digests may be produced by placing the tumor in enzymatic mediaand mechanically dissociating the tumor for approximately 1 minute,followed by incubation for 30 minutes at 37° C. in 5% CO₂, followed byrepeated cycles of mechanical dissociation and incubation under theforegoing conditions until only small tissue pieces are present. At theend of this process, if the cell suspension contains a large number ofred blood cells or dead cells, a density gradient separation usingFICOLL branched hydrophilic polysaccharide may be performed to removethese cells. Alternative methods known in the art may be used, such asthose described in U.S. Patent Application Publication No. 2012/0244133A1, the disclosure of which is incorporated by reference herein. Any ofthe foregoing methods may be used in any of the embodiments describedherein for methods of expanding TILs or methods treating a cancer.

In an embodiment, REP can be performed in a gas permeable containerusing the aAPCs of the present disclosure by any suitable method. Forexample, TILs can be rapidly expanded using non-specific T cell receptorstimulation in the presence of interleukin-2 (IL-2) or interleukin-15(IL-15). The non-specific T cell receptor stimulus can include, forexample, about 30 ng/mL of an anti-CD3 antibody, e.g. OKT-3, amonoclonal anti-CD3 antibody (commercially available from Ortho-McNeil,Raritan, N.J., USA or Miltenyi Biotech, Auburn, Calif., USA) or UHCT-1(commercially available from BioLegend, San Diego, Calif., USA). TILscan be rapidly expanded by further stimulation of the TILs in vitro withone or more antigens, including antigenic portions thereof, such asepitope(s), of the cancer, which can be optionally expressed from avector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide,e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), optionally inthe presence of a T cell growth factor, such as 300 IU/mL IL-2 or IL-15.Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2,tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenicportions thereof. TIL may also be rapidly expanded by re-stimulationwith the same antigen(s) of the cancer pulsed onto HLA-A2-expressingantigen-presenting cells. Alternatively, the TILs can be furtherre-stimulated with, e.g., example, irradiated, autologous lymphocytes orwith irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, a method for expanding TILs may include using about5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about10000 mL of cell culture medium, or about 5800 mL to about 8700 mL ofcell culture medium. In an embodiment, a method for expanding TILs mayinclude using about 1000 mL to about 2000 mL of cell medium, about 2000mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000mL of cell culture medium, about 4000 mL to about 5000 mL of cellculture medium, about 5000 mL to about 6000 mL of cell culture medium,about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL toabout 8000 mL of cell culture medium, about 8000 mL to about 9000 mL ofcell culture medium, about 9000 mL to about 10000 mL of cell culturemedium, about 10000 mL to about 15000 mL of cell culture medium, about15000 mL to about 20000 mL of cell culture medium, or about 20000 mL toabout 25000 mL of cell culture medium. In an embodiment, expanding thenumber of TILs uses no more than one type of cell culture medium. Anysuitable cell culture medium may be used, e.g., AIM-V cell medium(L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate)cell culture medium (Invitrogen, Carlsbad, Calif., USA). In this regard,the inventive methods advantageously reduce the amount of medium and thenumber of types of medium required to expand the number of TIL. In anembodiment, expanding the number of TIL may comprise feeding the cellsno more frequently than every third or fourth day. Expanding the numberof cells in a gas permeable container simplifies the proceduresnecessary to expand the number of cells by reducing the feedingfrequency necessary to expand the cells.

In an embodiment, the rapid expansion is performed using a gas permeablecontainer. Such embodiments allow for cell populations to expand fromabout 5×10⁵ cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In anembodiment, this expansion occurs without feeding. In an embodiment,this expansion occurs without feeding so long as medium resides at aheight of about 10 cm in a gas-permeable flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739 A1,International Patent Application Publication No. WO 2014/210036 A1, U.S.Patent Application Publication No. US 2013/0115617 A1, InternationalPublication No. WO 2013/188427 A1, U.S. Patent Application PublicationNo. US 2011/0136228 A1, U.S. Pat. No. 8,809,050, International PatentApplication Publication No. WO 2011/072088 A2, U.S. Patent ApplicationPublication No. US 2016/0208216 A1, U.S. Patent Application PublicationNo. US 2012/0244133 A1, International Patent Application Publication No.WO 2012/129201 A1, U.S. Patent Application Publication No. US2013/0102075 A1, U.S. Pat. No. 8,956,860, International PatentApplication Publication No. WO 2013/173835 A1, and U.S. PatentApplication Publication No. US 2015/0175966 A1, the disclosures of whichare incorporated herein by reference. Such processes are also describedin Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure ofwhich is incorporated by reference herein.

In an embodiment, the gas permeable container is a G-Rex 10 flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 10 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 40 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 100 to 300 million TILs after 2 mediumexchanges.

In an embodiment, the gas permeable container is a G-Rex 100 flask(Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 450 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs after 2 mediumexchanges.

In an embodiment, the gas permeable container is a G-Rex 100M flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 1000 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs without mediumexchange.

In an embodiment, the gas permeable container is a G-Rex 100 L flask(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a 100 cm² gas permeableculture surface. In an embodiment, the gas permeable container includesa 2000 mL cell culture medium capacity. In an embodiment, the gaspermeable container provides 1 to 3 billion TILs without mediumexchange.

In an embodiment, the gas permeable container is a G-Rex 24 well plate(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 2 cm² gas permeable culture surface. In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 8 mL cell culture medium capacity. In anembodiment, the gas permeable container provides 20 to 60 million cellsper well after 2 medium exchanges.

In an embodiment, the gas permeable container is a G-Rex 6 well plate(Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 10 cm² gas permeable culture surface. In anembodiment, the gas permeable container includes a plate with wells,wherein each well includes a 40 mL cell culture medium capacity. In anembodiment, the gas permeable container provides 100 to 300 millioncells per well after 2 medium exchanges.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, the rapid expansion uses about 1×10⁹ to about 1×10¹¹aAPCs. In an embodiment, the rapid expansion uses about 1×10⁹ aAPCs. Inan embodiment, the rapid expansion uses about 1×10¹⁰ aAPCs. In anembodiment, the rapid expansion uses about 1×10¹¹ aAPCs.

In an embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is selected fromthe group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40,1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100,1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150,1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200,1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450, and 1:500. In apreferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about1:90. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC)is about 1:95. In a preferred embodiment, the ratio of TILs to aAPCs(TIL:aAPC) is about 1:100. In a preferred embodiment, the ratio of TILsto aAPCs (TIL:aAPC) is about 1:105. In a preferred embodiment, the ratioof TILs to aAPCs (TIL:aAPC) is about 1:110.

In an embodiment, the ratio of TILs to aAPCs in the rapid expansion isabout 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250,about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratioof TILs to aAPCs in the rapid expansion is between 1 to 50 and 1 to 300.In an embodiment, the ratio of TILs to aAPCs in the rapid expansion isbetween 1 to 100 and 1 to 200.

In an embodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises an OKT-3 antibody.In a preferred embodiment, the cell culture medium comprises about 30ng/mL of OKT-3 antibody. In an embodiment, the cell culture mediumcomprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL,about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL,about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, andabout 1 μg/mL of OKT-3 antibody. In an embodiment, the cell culturemedium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL,between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3antibody.

In an embodiment, a rapid expansion process for TILs may be performedusing T-175 flasks and gas permeable bags as previously described (Tran,et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA). For TIL rapid expansion in T-175 flasks, 1×10⁶TILs suspended in 150 mL of media may be added to each T-175 flask. TheTILs may be cultured with aAPCs at a ratio of 1 TIL to 100 aAPCs and thecells were cultured in a 1 to 1 mixture of CM and AIM-V medium,supplemented with 3000 IU (international units) per mL of IL-2 and 30 ngper ml of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks may beincubated at 37° C. in 5% CO₂. Half the media may be exchanged on day 5using 50/50 medium with 3000 IU per mL of IL-2. On day 7 cells from twoT-175 flasks may be combined in a 3 liter bag and 300 mL of AIM V with5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml ofTIL suspension. The number of cells in each bag was counted every day ortwo and fresh media was added to keep the cell count between 0.5 and2.0×10⁶ cells/mL.

In an embodiment, for TIL rapid expansions in 500 mL capacity gaspermeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100,commercially available from Wilson Wolf Manufacturing Corporation, NewBrighton, Minn., USA), 5×10⁶ or 10×10⁶ TIL may be cultured with aAPCs ata ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5%human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3(OKT-3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO₂. Onday 5, 250 mL of supernatant may be removed and placed into centrifugebottles and centrifuged at 1500 rpm (revolutions per minute; 491×g) for10 minutes. The TIL pellets may be re-suspended with 150 mL of freshmedium with 5% human AB serum, 3000 IU per mL of IL-2, and added back tothe original G-Rex 100 flasks. When TIL are expanded serially in G-Rex100 flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the300 mL of media present in each flask and the cell suspension may bedivided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mLof IL-2 may be added to each flask. The G-Rex 100 flasks may beincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU per mL of IL-2 may be added to each G-Rex 100 flask. The cells may beharvested on day 14 of culture.

In an embodiment, TILs may be prepared as follows. 2 mm³ tumor fragmentsare cultured in complete media (CM) comprised of AIM-V medium(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 2 mMglutamine (Mediatech, Inc. Manassas, Va.), 100 U/mL penicillin(Invitrogen Life Technologies), 100 μg/mL streptomycin (Invitrogen LifeTechnologies), 5% heat-inactivated human AB serum (Valley Biomedical,Inc. Winchester, Va.) and 600 IU/mL rhlL-2 (Chiron, Emeryville, Calif.).For enzymatic digestion of solid tumors, tumor specimens were diced intoRPMI-1640, washed and centrifuged at 800 rpm for 5 minutes at 15-22° C.,and resuspended in enzymatic digestion buffer (0.2 mg/mL Collagenase and30 units/ml of DNase in RPMI-1640) followed by overnight rotation atroom temperature. TILs established from fragments may be grown for 3-4weeks in CM and expanded fresh or cryopreserved in heat-inactivated HABserum with 10% dimethylsulfoxide (DMSO) and stored at −180° C. until thetime of study. Tumor associated lymphocytes (TAL) obtained from ascitescollections were seeded at 3×10⁶ cells/well of a 24 well plate in CM.TIL growth was inspected about every other day using a low-powerinverted microscope.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. U.S. PatentApplication Publication No. 2005/0106717 A1, the disclosures of whichare incorporated herein by reference. In an embodiment, TILs areexpanded in gas-permeable bags. In an embodiment, TILs are expandedusing a cell expansion system that expands TILs in gas permeable bags,such as the Xuri Cell Expansion System W25 (GE Healthcare). In anembodiment, TILs are expanded using a cell expansion system that expandsTILs in gas permeable bags, such as the WAVE Bioreactor System, alsoknown as the Xuri Cell Expansion System W5 (GE Healthcare). In anembodiment, the cell expansion system includes a gas permeable cell bagwith a volume selected from the group consisting of about 100 mL, about200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L,about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, andabout 30 L. In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume range selected from the groupconsisting of between 50 and 150 mL, between 150 and 250 mL, between 250and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550and 650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850and 950 mL, and between 950 and 1050 mL. In an embodiment, the cellexpansion system includes a gas permeable cell bag with a volume rangeselected from the group consisting of between 1 L and 2 L, between 2 Land 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L,between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9L and 10 L, between 10 L and 11 L, between 11 L and 12 L, between 12 Land 13 L, between 13 L and 14 L, between 14 L and 15 L, between 15 L and16 L, between 16 L and 17 L, between 17 L and 18 L, between 18 L and 19L, and between 19 L and 20 L. In an embodiment, the cell expansionsystem includes a gas permeable cell bag with a volume range selectedfrom the group consisting of between 0.5 L and 5 L, between 5 L and 10L, between 10 L and 15 L, between 15 L and 20 L, between 20 L and 25 L,and between 25 L and 30 L. In an embodiment, the cell expansion systemutilizes a rocking time of about 30 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 24 hours, about 2 days, about 3 days, about 4days, about 5 days, about 6 days, about 7 days, about 8 days, about 9days, about 10 days, about 11 days, about 12 days, about 13 days, about14 days, about 15 days, about 16 days, about 17 days, about 18 days,about 19 days, about 20 days, about 21 days, about 22 days, about 23days, about 24 days, about 25 days, about 26 days, about 27 days, andabout 28 days. In an embodiment, the cell expansion system utilizes arocking time of between 30 minutes and 1 hour, between 1 hour and 12hours, between 12 hours and 1 day, between 1 day and 7 days, between 7days and 14 days, between 14 days and 21 days, and between 21 days and28 days. In an embodiment, the cell expansion system utilizes a rockingrate of about 2 rocks/minute, about 5 rocks/minute, about 10rocks/minute, about 20 rocks/minute, about 30 rocks/minute, and about 40rocks/minute. In an embodiment, the cell expansion system utilizes arocking rate of between 2 rocks/minute and 5 rocks/minute, 5rocks/minute and 10 rocks/minute, 10 rocks/minute and 20 rocks/minute,20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40rocks/minute. In an embodiment, the cell expansion system utilizes arocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In anembodiment, the cell expansion system utilizes a rocking angle ofbetween 2° and 3°, between 3° and 4°, between 4° and 5°, between 5° and6°, between 6° and 7°, between 7° and 8°, between 8° and 9°, between 9°and 10°, between 10° and 11°, and between 11° and 12°.

In an embodiment, a method of expanding TILs using aAPCs furthercomprises a step wherein TILs are selected for superior tumorreactivity. Any selection method known in the art may be used. Forexample, the methods described in U.S. Patent Application PublicationNo. 2016/0010058 A1, the disclosures of which are incorporated herein byreference, may be used for selection of TILs for superior tumorreactivity.

In an embodiment, the aAPCs of the present invention may be used toexpand T cells. Any of the foregoing embodiments of the presentinvention described for the expansion of TILs may also be applied to theexpansion of T cells. In an embodiment, the aAPCs of the presentinvention may be used to expand CD8⁺ T cells. In an embodiment, theaAPCs of the present invention may be used to expand CD4⁺ T cells. In anembodiment, the aAPCs of the present invention may be used to expand Tcells transduced with a chimeric antigen receptor (CAR-T). In anembodiment, the aAPCs of the present invention may be used to expand Tcells comprising a modified T cell receptor (TCR). The CAR-T cells maybe targeted against any suitable antigen, including CD19, as describedin the art, e.g., in U.S. Pat. Nos. 7,070,995; 7,446,190; 8,399,645;8,916,381; and 9,328,156; the disclosures of which are incorporated byreference herein. The modified TCR cells may be targeted against anysuitable antigen, including NY-ESO-1, TRP-1, TRP-2, tyrosinase cancerantigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof, asdescribed in the art, e.g., in U.S. Pat. Nos. 8,367,804 and 7,569,664,the disclosures of which are incorporated by reference herein.

Methods of Treating Cancers and Other Diseases

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinghyperproliferative disorders. They may also be used in treating otherdisorders as described herein and in the following paragraphs. The TILs,populations and compositions thereof described herein may be for use inthe treatment of a disease. In an embodiment, the TILs, populations andcompositions described herein are for use in the treatment of ahyperproliferative disorder.

In some embodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of melanoma, ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer, renal cancer, andrenal cell carcinoma, pancreatic cancer, and glioblastoma. In someembodiments, the hyperproliferative disorder is a hematologicalmalignancy. In some embodiments, the hematological malignancy isselected from the group consisting of chronic lymphocytic leukemia,acute lymphoblastic leukemia, diffuse large B cell lymphoma,non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, andmantle cell lymphoma.

In an embodiment, the invention includes a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of: (a) obtaining a first population of TILs from a tumorresected from a patient; (b) performing a rapid expansion of the firstpopulation of TILs using a population of artificial antigen presentingcells (aAPCs) in a cell culture medium to obtain a second population ofTILs, wherein the second population of TILs is at least 50-fold greaterin number than the first population of TILs; and (c) administering atherapeutically effective portion of the second population of TILs to apatient with the cancer. In an embodiment, the aAPCs comprise MOLM-14cells transduced with one or more viral vectors, wherein the one or moreviral vectors comprise a nucleic acid encoding CD86 and a nucleic acidencoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 proteinand a 4-1BBL protein. In an embodiment, the rapid expansion is performedover a period not greater than 14 days.

In an embodiment, the invention includes a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of: (a) obtaining a first population of TILs from a tumorresected from a patient; (b) performing an initial expansion of thefirst population of TILs using a first population of artificial antigenpresenting cells (aAPCs) in a first cell culture medium to obtain asecond population of TILs, wherein the second population of TILs is atleast 10-fold greater in number than the first population of TILs, andwherein the first cell culture medium comprises IL-2; (c) performing arapid expansion of the second population of TILs using a secondpopulation of aAPCs in a second cell culture medium to obtain a thirdpopulation of TILs, wherein the third population of TILs is at least50-fold greater in number than the first population of TILs; and whereinthe second cell culture medium comprises IL-2 and OKT-3; (d)administering a therapeutically effective portion of the thirdpopulation of TILs to a patient with the cancer. In an embodiment, theaAPCs comprise MOLM-14 cells transduced with one or more viral vectors,wherein the one or more viral vectors comprise a nucleic acid encodingCD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cellsexpress a CD86 protein and a 4-1BBL protein. In an embodiment, the rapidexpansion is performed over a period not greater than 14 days. In anembodiment, the initial expansion is performed using a gas permeablecontainer.

In an embodiment, the invention includes a method of treating a cancerwith a population of tumor infiltrating lymphocytes (TILs) comprisingthe steps of: (a) obtaining a first population of TILs from a tumorresected from a patient; (b) performing an initial expansion of thefirst population of TILs in a first cell culture medium to obtain asecond population of TILs, wherein the second population of TILs is atleast 10-fold greater in number than the first population of TILs, andwherein the first cell culture medium comprises IL-2; (c) performing arapid expansion of the second population of TILs using a population ofartificial antigen presenting cells (aAPCs) in a second cell culturemedium to obtain a third population of TILs, wherein the thirdpopulation of TILs is at least 50-fold greater in number than the firstpopulation of TILs; and wherein the second cell culture medium comprisesIL-2 and OKT-3; (d) administering a therapeutically effective portion ofthe third population of TILs to a patient with the cancer. In anembodiment, the aAPCs comprise MOLM-14 cells transduced with one or moreviral vectors, wherein the one or more viral vectors comprise a nucleicacid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein theMOLM-14 cells express a CD86 protein and a 4-1BBL protein. In anembodiment, the rapid expansion is performed over a period not greaterthan 14 days.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the non-myeloablativechemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26prior to TIL infusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to23 prior to TIL infusion). In an embodiment, after non-myeloablativechemotherapy and TIL infusion (at day 0) according to the presentdisclosure, the patient receives an intravenous infusion of IL-2intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Efficacy of the compounds and combinations of compounds described hereinin treating, preventing and/or managing the indicated diseases ordisorders can be tested using various models known in the art, whichprovide guidance for treatment of human disease. For example, models fordetermining efficacy of treatments for ovarian cancer are described,e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, etal., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy oftreatments for pancreatic cancer are described in Herreros-Villanueva,et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models fordetermining efficacy of treatments for breast cancer are described,e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models fordetermining efficacy of treatments for melanoma are described, e.g., inDamsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Modelsfor determining efficacy of treatments for lung cancer are described,e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664.Models for determining efficacy of treatments for lung cancer aredescribed, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60;and Sano, Head Neck Oncol. 2009, 1, 32.

Non-Myeloablative Lymphodepletion with Chemotherapy

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the invention provides apopulation of TILs obtainable by a method described herein for use intreating a cancer, wherein the population of TILs is for treating apatient which is pre-treated with non-myeloablative chemotherapy. In anembodiment, the non-myeloablative chemotherapy is cyclophosphamide 60mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) andfludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior to TIL infusion).In an embodiment, after non-myeloablative chemotherapy and TIL infusion(at day 0) according to the present disclosure, the patient receives anintravenous infusion of IL-2 (aldesleukin, commercially available asPROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologictolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the aAPC-expanded TILs of the invention.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabineis administered at a concentration of 1 μg/mL fludarabine. In someembodiments, the fludarabine treatment is administered for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/ml-10 μg/mlby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m²/day, 150mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day,275 mg/m²/day, or 300 mg/m²/day. In some embodiments, thecyclophosphamide is administered intravenously (i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m²/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide are together to a patient. In someembodiments, fludarabine is administered at 25 mg/m²/day i.v. andcyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m²/day for two days followed byadministration of fludarabine at a dose of 25 mg/m²/day for five days.

Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using aAPCs of the present disclosureare administered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using aAPCs of the present disclosure maybe administered by any suitable route as known in the art. Preferably,the TILs are administered as a single infusion, such as anintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. Preferably, from about2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an average ofaround 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of theTILs provided in the pharmaceutical compositions of the invention is inthe range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰,1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of embodiments ofthe invention are effective over a wide dosage range. The exact dosagewill depend upon the route of administration, the form in which thecompound is administered, the gender and age of the subject to betreated, the body weight of the subject to be treated, and thepreference and experience of the attending physician. Theclinically-established dosages of the TILs may also be used ifappropriate. The amounts of the pharmaceutical compositions administeredusing the methods herein, such as the dosages of TILs, will be dependenton the human or mammal being treated, the severity of the disorder orcondition, the rate of administration, the disposition of the activepharmaceutical ingredients and the discretion of the prescribingphysician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³,5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, aneffective dosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹,1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹,5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1—Variability in Expansion of Tumor Infiltrating LymphocytesUsing PBMC Feeder Cells

The variability in TIL expansion obtained by use of PBMC feeder cellsmay be demonstrated by comparing the results of multiple TIL expansionson the same line of TILs obtained from a patient. FIG. 1 illustratestypical results of rapid expansion of TILs using irradiated allogeneicPBMC feeder cells (PBMC feeders). Two TIL lines labeled M1015T andM1016T (1.3×10⁵ cells) were co-cultured with 46 different irradiatedfeeder cell lots (1.3×10⁷), IL-2 (3000 IU/mL, recombinant human IL-2(e.g., aldesleukin or equivalent), CellGenix, Inc., Portsmouth, N.H.,USA) and OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH,Bergisch Gladbach, Germany) in a T25 flask for 7 days. The foldexpansion value for TILs was calculated on Day 7. The figure shows thenumber of fold expansions for the two TIL lines in separate stimulationexperiments. For each TIL line, 46 different PBMC feeder lots weretested. The results range over more than 100-fold for each TIL line, andhighlight the variability of expansion results using PBMC feeder cells.The aAPCs of the present invention offer reduced variability inexpansion performance compared to PBMC feeders, as well as otheradvantages, as shown in the following examples.

Example 2—Selection of Myeloid Cells for aAPC Development

Phenotypic characterization was performed on various myeloid-lineagecell lines to identify potential candidates for further modificationinto aAPCs for TIL expansion. The results are summarized in Table 5. TheMOLM-14 cell line exhibited endogenous expression of CD64, and wasselected for further development. The EM-3 cell line was selected basedon the observation of endogenous expression of ICOS-L (which was notobserved for the EM-2 cell line, despite being taken from the samepatient).

TABLE 5 Summary of costimulatory molecules expressed endogenously oncandidate cell lines for aAPCs. CML refers to chronic myeloid leukemia,and AML refers to acute myeloid leukemia. “Pop” refers to the populationof cells observed to express the marker (½ pop = 50%). Cell line EM-2EM-3 K562 Myeloid blast Myeloid blast KG1-246 KG1-8031 myeloid erythro-MOLM-14 Origin crisis, CML crisis, CML AML AML leukemia, CML AMLHLA-A/B/C + + + + − + CD64 − − − − − + CD80 − − − − − + ICOS-L − + − −− + 4-1BBL − − − − − − PD-L1 − − − − − − CD58 + + + + + + CD86 − − − −− + (½ pop)

Example 3—Preparation of MOLM-14 Artificial Antigen Presenting Cells(aMOLM14 aAPCs)

MOLM-14 cells were obtained from Leibniz-Institut DSMZ-Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH. To develop MOLM-14 basedaAPCs, MOLM-14 cells were engineered with the costimulatory moleculesCD86 and 4-1BBL (CD137L). Human CD86 (hCD86) and human 4-1BBL (h4-1BBL)genes were cloned into commercially-available PLV430G and co-transfectedwith PDONR221 vectors (Invitrogen/Thermo Fisher Scientific, Carlsbad,Calif., USA) using a lentiviral transduction method. The gateway cloningmethod was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4,571-589, to clone hCD86 and hCD137L genes onto the PLV430G and PDONR221vectors. The 293T cell line (human embryonic kidney cells transformedwith large T antigen) was used for lentiviral production, transduced toMOLM-14 cells. The transfected cells were sorted (S3e Cell Sorter,Bio-Rad, Hercules, Calif., USA) using APC-conjugated CD86 andPE-conjugated CD137L to isolate and enrich the cells. The enriched cellswere checked for purity by flow cytometry.

The vectors and portions thereof used for cloning are depicted in FIG. 2to FIG. 11, and the nucleotide sequences for each vector are given inTable 6. The pLV430G human 4-1BBL vector is illustrated in FIG. 2, withthe polymerase chain reaction product (PCRP) portion shown in FIG. 3.The pLV430G human CD86 vector is illustrated in FIG. 4, with the PCRPportion shown in FIG. 5. The pDONR221 human CD86 donor and human 4-1BBLdonor vectors are shown in FIG. 6 and FIG. 7, respectively. Diagrams ofthe empty pLV430G destination vector and empty pDONR221 donor vector forthe Gateway cloning method are shown in FIG. 8 and FIG. 9, respectively.FIG. 10 and FIG. 11 illustrate vector diagrams of the psPAX2 andpCIGO-VSV.G helper plasmids used for lentivirus production.

TABLE 6Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.Identifier (Description) Sequence SEQ ID NO: 15cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(pLV430G humangttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 1204-1BBL vector)cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700caacaagttt gtacaaaaaa gcaggcttcg ccaccatgga atacgcctct gatgccagcc 2760tggaccccga agctccttgg cctcctgccc ctagagccag agcctgtaga gtgctgcctt 2820gggctctggt ggctggcctt ctccttctgc tgctgctggc cgctgcctgc gctgtgtttc 2880tggcttgtcc ttgggccgtg tcaggcgcca gagcttctcc tggatctgcc gccagcccca 2940gactgagaga gggacctgag ctgagccccg atgatcctgc cggactgctg gatctgagac 3000agggcatgtt cgcccagctg gtggcccaga acgtgctgct gatcgatggc cccctgagct 3060ggtacagcga tcctggactg gctggcgtgt cactgacagg cggcctgagc tacaaagagg 3120acaccaaaga actggtggtg gccaaggccg gcgtgtacta cgtgttcttt cagctggaac 3180tgcggagagt ggtggccggc gaaggatccg gctctgtgtc tctggcactg catctgcagc 3240ccctgagatc tgctgcaggc gctgctgcac tggccctgac agtggacctg cctccagcct 3300ctagcgaggc cagaaactcc gcattcgggt ttcaaggcag actgctgcac ctgtctgccg 3360gccagagact gggagtgcat ctgcacacag aggccagagc cagacacgcc tggcagctga 3420cacagggcgc tacagtgctg ggcctgttca gagtgacccc cgaaattcca gccggcctgc 3480ccagccctag aagcgagtag gacccagctt tcttgtacaa agtggtgatt cgagttaatt 3540aagctagcct agtgccattt gttcagtggt tcgtagggct ttcccccact gtttggcttt 3600cagttatatg gatgatgtgg tattgggggc caagtctgta cagcatcttg agtccctttt 3660taccgctgtt accaattttc ttttgtcttt gggtatacat ttaaacccta acaaaacaaa 3720gagatggggt tactctctaa attttatggg ttatgtcatt ggatgttatg ggtccttgcc 3780acaagaacac atcatacaaa aaatcaaaga atgttttaga aaacttccta ttaacaggcc 3840tattgattgg aaagtatgtc aacgaattgt gggtcttttg ggttttgctg ccccttttac 3900acaatgtggt tatcctgcgt tgatgccttt gtatgcatgt attcaatcta agcaggcttt 3960cactttctcg ccaacttaca aggcctttct gtgtaaacaa tacctgaacc tttaccccgt 4020tgcccggcaa cggccaggtc tgtgccaagt gtttgctgac gcaaccccca ctggctgggg 4080cttggtcatg ggccatcagc gcatgcgtgg aaccttttcg gctcctctgc cgatccatac 4140tgcggaactc ctagccgctt gttttgctcg cagcaggtct ggagcaaaca ttatcgggac 4200tgataactct gttgtcctat cccgcaaata tacatcgttt ccatggctgc taggctgtgc 4260tgccaactgg atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc 4320ggacgaccct tctcggggtc gcttgggact ctctcgtccc cttctccgtc tgccgttccg 4380accgaccacg gggcgcacct ctctttacgc ggactccccg tctgtgcctt ctcatctgcc 4440ggaccgtgtg cacttcgctt cacctctgca cgtcgcatgg agaccaccgt gaacgcccac 4500caaatattgc ccaaggtctt acataagagg actcttggac tctcagcaat gtcaacgacc 4560gaccttgagg catacttcaa agactgtttg tttaaagact gggaggagtt gggggaggag 4620attaggttaa aggtctttgt actaggaggc tgtaggcata aattggtctg cgcaccagca 4680ccatggcgca atcactagag cggggtacct ttaagaccaa tgacttacaa ggcagctgta 4740gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga 4800agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg 4860gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 4920cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 4980ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat 5040ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat 5100aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 5160cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta 5220tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 5280cccatggctg actaattttt tttatttatg cagaggccga ggccggatcc cttgagtggc 5340tttcatcctg gagcagactt tgcagtctgt ggactgcaac acaacattgc ctttatgtgt 5400aactcttggc tgaagctctt acaccaatgc tgggggacat gtacctccca ggggcccagg 5460aagactacgg gaggctacac caacgtcaat cagaggggcc tgtgtagcta ccgataagcg 5520gaccctcaag agggcattag caatagtgtt tataaggccc ccttgttaat tcttgaagac 5580gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 5640agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 5700aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 5760attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5820cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5880aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5940ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6000gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact 6060attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 6120tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 6180tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 6240atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 6300agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg 6360aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 6420caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 6480ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 6540gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6600tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 6660atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6720tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6780accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6840gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6900caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 6960tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7020ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7080tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 7140gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 7200attgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 7260gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 7320gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 7380ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 7440ggcctttttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 7500acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 7560ctcgcgtcg 7569 SEQ ID NO: 16atggaatacg cctctgatgc cagcctggac cccgaagctc cttggcctcc tgcccctaga 60(4-1BBL CoOP)gccagagcct gtagagtgct gccttgggct ctggtggctg gccttctcct tctgctgctg 120ctggccgctg cctgcgctgt gtttctggct tgtccttggg ccgtgtcagg cgccagagct 180tctcctggat ctgccgccag ccccagactg agagagggac ctgagctgag ccccgatgat 240cctgccggac tgctggatct gagacagggc atgttcgccc agctggtggc ccagaacgtg 300ctgctgatcg atggccccct gagctggtac agcgatcctg gactggctgg cgtgtcactg 360acaggcggcc tgagctacaa agaggacacc aaagaactgg tggtggccaa ggccggcgtg 420tactacgtgt tctttcagct ggaactgcgg agagtggtgg ccggcgaagg atccggctct 480gtgtctctgg cactgcatct gcagcccctg agatctgctg caggcgctgc tgcactggcc 540ctgacagtgg acctgcctcc agcctctagc gaggccagaa actccgcatt cgggtttcaa 600ggcagactgc tgcacctgtc tgccggccag agactgggag tgcatctgca cacagaggcc 660agagccagac acgcctggca gctgacacag ggcgctacag tgctgggcct gttcagagtg 720acccccgaaa ttccagccgg cctgcccagc cctagaagcg agtag 765 SEQ ID NO: 17ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg gaatacgcct ctgatgccag 60(4-1BBL PRCP)cctggacccc gaagctcctt ggcctcctgc ccctagagcc agagcctgta gagtgctgcc 120ttgggctctg gtggctggcc ttctccttct gctgctgctg gccgctgcct gcgctgtgtt 180tctggcttgt ccttgggccg tgtcaggcgc cagagcttct cctggatctg ccgccagccc 240cagactgaga gagggacctg agctgagccc cgatgatcct gccggactgc tggatctgag 300acagggcatg ttcgcccagc tggtggccca gaacgtgctg ctgatcgatg gccccctgag 360ctggtacagc gatcctggac tggctggcgt gtcactgaca ggcggcctga gctacaaaga 420ggacaccaaa gaactggtgg tggccaaggc cggcgtgtac tacgtgttct ttcagctgga 480actgcggaga gtggtggccg gcgaaggatc cggctctgtg tctctggcac tgcatctgca 540gcccctgaga tctgctgcag gcgctgctgc actggccctg acagtggacc tgcctccagc 600ctctagcgag gccagaaact ccgcattcgg gtttcaaggc agactgctgc acctgtctgc 660cggccagaga ctgggagtgc atctgcacac agaggccaga gccagacacg cctggcagct 720gacacagggc gctacagtgc tgggcctgtt cagagtgacc cccgaaattc cagccggcct 780gcccagccct agaagcgagt aggacccagc tttcttgtac aaagtggtcc cc 832SEQ ID NO: 18cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(pLV430G humangttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120CD86 vector)cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700caacaagttt gtacaaaaaa gcaggcttcg ccaccatggg cctgagcaac atcctgttcg 2760tgatggcctt cctgctgtcc ggagccgccc ctctgaagat ccaggcctac ttcaacgaga 2820ccgccgacct gccctgccag ttcgccaaca gccagaacca gagcctgagc gaactggtgg 2880tgttctggca ggaccaggaa aacctggtcc tgaacgaggt gtacctgggc aaagaaaagt 2940tcgacagcgt gcacagcaag tacatgggcc ggaccagctt cgacagcgac agctggaccc 3000tgcggctgca caacctgcag atcaaggaca agggcctgta ccagtgcatc atccaccaca 3060agaaacccac cggcatgatc agaatccacc agatgaacag cgagctgtcc gtgctggcca 3120acttcagcca gcccgagatc gtgcccatca gcaacatcac cgagaacgtg tacatcaacc 3180tgacctgcag cagcatccac ggctaccccg agcccaagaa aatgagcgtg ctgctgcgga 3240ccaagaacag caccatcgag tacgacggcg tgatgcagaa aagccaggac aacgtgaccg 3300agctgtacga cgtgagcatc agcctgagcg tgagcttccc cgacgtgacc agcaacatga 3360ccatcttttg catcctggaa accgacaaga cccggctgct gtccagcccc ttcagcatcg 3420agctggaaga tccccagccc cctcccgacc acatcccctg gatcaccgcc gtgctgccca 3480ccgtgatcat ctgcgtgatg gtgttctgcc tgatcctgtg gaagtggaag aagaagaagc 3540ggcctaggaa cagctacaag tgcggcacca acaccatgga acgggaggaa agcgagcaga 3600ccaagaagcg ggagaagatc cacatccccg agcggagcga cgaggcccag cgggtgttca 3660agaggagcaa gaccagcagc tgcgacaaga gcgacacctg cttctaggac ccagctttct 3720tgtacaaagt ggtgattcga gttaattaag ctagcctagt gccatttgtt cagtggttcg 3780tagggctttc ccccactgtt tggctttcag ttatatggat gatgtggtat tgggggccaa 3840gtctgtacag catcttgagt ccctttttac cgctgttacc aattttcttt tgtctttggg 3900tatacattta aaccctaaca aaacaaagag atggggttac tctctaaatt ttatgggtta 3960tgtcattgga tgttatgggt ccttgccaca agaacacatc atacaaaaaa tcaaagaatg 4020ttttagaaaa cttcctatta acaggcctat tgattggaaa gtatgtcaac gaattgtggg 4080tcttttgggt tttgctgccc cttttacaca atgtggttat cctgcgttga tgcctttgta 4140tgcatgtatt caatctaagc aggctttcac tttctcgcca acttacaagg cctttctgtg 4200taaacaatac ctgaaccttt accccgttgc ccggcaacgg ccaggtctgt gccaagtgtt 4260tgctgacgca acccccactg gctggggctt ggtcatgggc catcagcgca tgcgtggaac 4320cttttcggct cctctgccga tccatactgc ggaactccta gccgcttgtt ttgctcgcag 4380caggtctgga gcaaacatta tcgggactga taactctgtt gtcctatccc gcaaatatac 4440atcgtttcca tggctgctag gctgtgctgc caactggatc ctgcgcggga cgtcctttgt 4500ttacgtcccg tcggcgctga atcctgcgga cgacccttct cggggtcgct tgggactctc 4560tcgtcccctt ctccgtctgc cgttccgacc gaccacgggg cgcacctctc tttacgcgga 4620ctccccgtct gtgccttctc atctgccgga ccgtgtgcac ttcgcttcac ctctgcacgt 4680cgcatggaga ccaccgtgaa cgcccaccaa atattgccca aggtcttaca taagaggact 4740cttggactct cagcaatgtc aacgaccgac cttgaggcat acttcaaaga ctgtttgttt 4800aaagactggg aggagttggg ggaggagatt aggttaaagg tctttgtact aggaggctgt 4860aggcataaat tggtctgcgc accagcacca tggcgcaatc actagagcgg ggtaccttta 4920agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 4980ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 5040ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 5100aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 5160tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt 5220agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt 5280gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 5340ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 5400gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa 5460ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 5520aggccgaggc cggatccctt gagtggcttt catcctggag cagactttgc agtctgtgga 5580ctgcaacaca acattgcctt tatgtgtaac tcttggctga agctcttaca ccaatgctgg 5640gggacatgta cctcccaggg gcccaggaag actacgggag gctacaccaa cgtcaatcag 5700aggggcctgt gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat 5760aaggccccct tgttaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg 5820ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 5880gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 5940aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 6000tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 6060aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 6120aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 6180tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 6240aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 6300tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 6360ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 6420taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 6480agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa 6540caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 6600tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 6660gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 6720cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 6780caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 6840ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 6900aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 6960gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7020atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 7080tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 7140gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 7200actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 7260gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 7320agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 7380ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa 7440aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 7500cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 7560gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 7620cctttttacg gttcctggcc ttttgctggc ctttttgaag ctgtccctga tggtcgtcat 7680ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa 7740gaatcataat ggggaaggcc atccagcctc gcgtcg 7776 SEQ ID NO: 19atgggcctga gcaacatcct gttcgtgatg gccttcctgc tgtccggagc cgcccctctg 60(CD86 CoOP)aagatccagg cctacttcaa cgagaccgcc gacctgccct gccagttcgc caacagccag 120aaccagagcc tgagcgaact ggtggtgttc tggcaggacc aggaaaacct ggtcctgaac 180gaggtgtacc tgggcaaaga aaagttcgac agcgtgcaca gcaagtacat gggccggacc 240agcttcgaca gcgacagctg gaccctgcgg ctgcacaacc tgcagatcaa ggacaagggc 300ctgtaccagt gcatcatcca ccacaagaaa cccaccggca tgatcagaat ccaccagatg 360aacagcgagc tgtccgtgct ggccaacttc agccagcccg agatcgtgcc catcagcaac 420atcaccgaga acgtgtacat caacctgacc tgcagcagca tccacggcta ccccgagccc 480aagaaaatga gcgtgctgct gcggaccaag aacagcacca tcgagtacga cggcgtgatg 540cagaaaagcc aggacaacgt gaccgagctg tacgacgtga gcatcagcct gagcgtgagc 600ttccccgacg tgaccagcaa catgaccatc ttttgcatcc tggaaaccga caagacccgg 660ctgctgtcca gccccttcag catcgagctg gaagatcccc agccccctcc cgaccacatc 720ccctggatca ccgccgtgct gcccaccgtg atcatctgcg tgatggtgtt ctgcctgatc 780ctgtggaagt ggaagaagaa gaagcggcct aggaacagct acaagtgcgg caccaacacc 840atggaacggg aggaaagcga gcagaccaag aagcgggaga agatccacat ccccgagcgg 900agcgacgagg cccagcgggt gttcaagagc agcaagacca gcagctgcga caagagcgac 960acctgcttc 969 SEQ ID NO: 20ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg ggcctgagca acatcctgtt 60(CD86 PCRP)cgtgatggcc ttcctgctgt ccggagccgc ccctctgaag atccaggcct acttcaacga 120gaccgccgac ctgccctgcc agttcgccaa cagccagaac cagagcctga gcgaactggt 180ggtgttctgg caggaccagg aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa 240gttcgacagc gtgcacagca agtacatggg ccggaccagc ttcgacagcg acagctggac 300cctgcggctg cacaacctgc agatcaagga caagggcctg taccagtgca tcatccacca 360caagaaaccc accggcatga tcagaatcca ccagatgaac agcgagctgt ccgtgctggc 420caacttcagc cagcccgaga tcgtgcccat cagcaacatc accgagaacg tgtacatcaa 480cctgacctgc agcagcatcc acggctaccc cgagcccaag aaaatgagcg tgctgctgcg 540gaccaagaac agcaccatcg agtacgacgg cgtgatgcag aaaagccagg acaacgtgac 600cgagctgtac gacgtgagca tcagcctgag cgtgagcttc cccgacgtga ccagcaacat 660gaccatcttt tgcatcctgg aaaccgacaa gacccggctg ctgtccagcc ccttcagcat 720cgagctggaa gatccccagc cccctcccga ccacatcccc tggatcaccg ccgtgctgcc 780caccgtgatc atctgcgtga tggtgttctg cctgatcctg tggaagtgga agaagaagaa 840gcggcctagg aacagctaca agtgcggcac caacaccatg gaacgggagg aaagcgagca 900gaccaagaag cgggagaaga tccacatccc cgagcggagc gacgaggccc agcgggtgtt 960caagagcagc aagaccagca gctgcgacaa gagcgacacc tgcttctagg acccagcttt 1020cttgtacaaa gtggtcccc 1039 SEQ ID NO: 21ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60(pDONR221 CD86taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120vector)gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa 660aagcaggctt cgccaccatg ggcctgagca acatcctgtt cgtgatggcc ttcctgctgt 720ccggagccgc ccctctgaag atccaggcct acttcaacga gaccgccgac ctgccctgcc 780agttcgccaa cagccagaac cagagcctga gcgaactggt ggtgttctgg caggaccagg 840aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa gttcgacagc gtgcacagca 900agtacatggg ccggaccagc ttcgacagcg acagctggac cctgcggctg cacaacctgc 960agatcaagga caagggcctg taccagtgca tcatccacca caagaaaccc accggcatga 1020tcagaatcca ccagatgaac agcgagctgt ccgtgctggc caacttcagc cagcccgaga 1080tcgtgcccat cagcaacatc accgagaacg tgtacatcaa cctgacctgc agcagcatcc 1140acggctaccc cgagcccaag aaaatgagcg tgctgctgcg gaccaagaac agcaccatcg 1200agtacgacgg cgtgatgcag aaaagccagg acaacgtgac cgagctgtac gacgtgagca 1260tcagcctgag cgtgagcttc cccgacgtga ccagcaacat gaccatcttt tgcatcctgg 1320aaaccgacaa gacccggctg ctgtccagcc ccttcagcat cgagctggaa gatccccagc 1380cccctcccga ccacatcccc tggatcaccg ccgtgctgcc caccgtgatc atctgcgtga 1440tggtgttctg cctgatcctg tggaagtgga agaagaagaa gcggcctagg aacagctaca 1500agtgcggcac caacaccatg gaacgggagg aaagcgagca gaccaagaag cgggagaaga 1560tccacatccc cgagcggagc gacgaggccc agcgggtgtt caagagcagc aagaccagca 1620gctgcgacaa gagcgacacc tgcttctagg acccagcttt cttgtacaaa gtggtcatta 1680taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa 1740tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg 1800tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa 1860taatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta 1920tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt 1980tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct 2040tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca 2100atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga 2160ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg 2220gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg 2280cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca 2340gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg 2400cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc 2460ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata 2520accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg 2580cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat 2640tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt 2700ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc 2760agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga 2820gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 2880atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 2940tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3000gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 3060actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 3120gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 3180agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 3240ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 3300aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 3360cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 3420gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 3480cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt 3526 SEQ ID NO: 22ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60(pDONR221 4-taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1201BBL vector)gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa 660aagcaggctt cgccaccatg gaatacgcct ctgatgccag cctggacccc gaagctcctt 720ggcctcctgc ccctagagcc agagcctgta gagtgctgcc ttgggctctg gtggctggcc 780ttctccttct gctgctgctg gccgctgcct gcgctgtgtt tctggcttgt ccttgggccg 840tgtcaggcgc cagagcttct cctggatctg ccgccagccc cagactgaga gagggacctg 900agctgagccc cgatgatcct gccggactgc tggatctgag acagggcatg ttcgcccagc 960tggtggccca gaacgtgctg ctgatcgatg gccccctgag ctggtacagc gatcctggac 1020tggctggcgt gtcactgaca ggcggcctga gctacaaaga ggacaccaaa gaactggtgg 1080tggccaaggc cggcgtgtac tacgtgttct ttcagctgga actgcggaga gtggtggccg 1140gcgaaggatc cggctctgtg tctctggcac tgcatctgca gcccctgaga tctgctgcag 1200gcgctgctgc actggccctg acagtggacc tgcctccagc ctctagcgag gccagaaact 1260ccgcattcgg gtttcaaggc agactgctgc acctgtctgc cggccagaga ctgggagtgc 1320atctgcacac agaggccaga gccagacacg cctggcagct gacacagggc gctacagtgc 1380tgggcctgtt cagagtgacc cccgaaattc cagccggcct gcccagccct agaagcgagt 1440aggacccagc tttcttgtac aaagtggtca ttataagaaa gcattgctta tcaatttgtt 1500gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgatatcc 1560cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc 1620aaaatctctg atgttacatt gcacaagata aaataatatc atcatgaaca ataaaactgt 1680ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga 1740ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata 1800atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt 1860tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac 1920taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg 1980atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag 2040aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc 2100attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg 2160cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg 2220gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt 2280cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa 2340taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc 2400tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg 2460gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct 2520aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact tgaggggagg 2580gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag 2640accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2700gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2760caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 2820tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2880ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2940tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3000gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3060tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3120gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3180gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3240ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3300ggccttttgc tcacatgtt 3319 SEQ ID NO: 23cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(pLV430Ggttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120vector)cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt 3600ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt 3660ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc 3720agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt 3780caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga 3840acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa 3900cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata 3960aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg 4020ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg 4080aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg 4140ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg 4200acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac 4260acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta 4320gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct 4380cgttcagctt tcttgtacaa agtggtgatt cgagttaatt aagctagcct agtgccattt 4440gttcagtggt tcgtagggct ttcccccact gtttggcttt cagttatatg gatgatgtgg 4500tattgggggc caagtctgta cagcatcttg agtccctttt taccgctgtt accaattttc 4560ttttgtcttt gggtatacat ttaaacccta acaaaacaaa gagatggggt tactctctaa 4620attttatggg ttatgtcatt ggatgttatg ggtccttgcc acaagaacac atcatacaaa 4680aaatcaaaga atgttttaga aaacttccta ttaacaggcc tattgattgg aaagtatgtc 4740aacgaattgt gggtcttttg ggttttgctg ccccttttac acaatgtggt tatcctgcgt 4800tgatgccttt gtatgcatgt attcaatcta agcaggcttt cactttctcg ccaacttaca 4860aggcctttct gtgtaaacaa tacctgaacc tttaccccgt tgcccggcaa cggccaggtc 4920tgtgccaagt gtttgctgac gcaaccccca ctggctgggg cttggtcatg ggccatcagc 4980gcatgcgtgg aaccttttcg gctcctctgc cgatccatac tgcggaactc ctagccgctt 5040gttttgctcg cagcaggtct ggagcaaaca ttatcgggac tgataactct gttgtcctat 5100cccgcaaata tacatcgttt ccatggctgc taggctgtgc tgccaactgg atcctgcgcg 5160ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc ggacgaccct tctcggggtc 5220gcttgggact ctctcgtccc cttctccgtc tgccgttccg accgaccacg gggcgcacct 5280ctctttacgc ggactccccg tctgtgcctt ctcatctgcc ggaccgtgtg cacttcgctt 5340cacctctgca cgtcgcatgg agaccaccgt gaacgcccac caaatattgc ccaaggtctt 5400acataagagg actcttggac tctcagcaat gtcaacgacc gaccttgagg catacttcaa 5460agactgtttg tttaaagact gggaggagtt gggggaggag attaggttaa aggtctttgt 5520actaggaggc tgtaggcata aattggtctg cgcaccagca ccatggcgca atcactagag 5580cggggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 5640agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc 5700ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 5760gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 5820tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 5880ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg 5940aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa 6000tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 6060caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc 6120atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 6180tttatttatg cagaggccga ggccggatcc cttgagtggc tttcatcctg gagcagactt 6240tgcagtctgt ggactgcaac acaacattgc ctttatgtgt aactcttggc tgaagctctt 6300acaccaatgc tgggggacat gtacctccca ggggcccagg aagactacgg gaggctacac 6360caacgtcaat cagaggggcc tgtgtagcta ccgataagcg gaccctcaag agggcattag 6420caatagtgtt tataaggccc ccttgttaat tcttgaagac gaaagggcct cgtgatacgc 6480ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6540cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 6600ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 6660agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 6720tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 6780gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 6840gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 6900gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6960gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 7020agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 7080ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 7140cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 7200gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 7260cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 7320gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 7380ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 7440acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 7500ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 7560aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 7620aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 7680ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 7740ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 7800actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 7860caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 7920gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7980ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 8040cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag cgccacgctt 8100cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 8160acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 8220ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 8280gccagcaacg cggccttttt acggttcctg gccttttgct ggcctttttg aagctgtccc 8340tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg 8400ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcg 8449 SEQ ID NO: 24ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60(pDONR221taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120vector)gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa 720aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt 780agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct 840gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca 900gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg 960tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt 1020catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta 1080caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt 1140ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat 1200attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca 1260gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc 1320cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc 1380agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc 1440tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc 1500atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac 1560ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc 1620attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac 1680tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg 1740acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat 1800acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccgccct 1860gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac 1920agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat 1980atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa 2040aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt 2100tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa 2160actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat 2220ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg 2280ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat 2340aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg 2400tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc 2460attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag 2520ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt 2580gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg 2640gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca 2700caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc gactacaggt 2760cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt ttacagtatt 2820atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg 2880tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt 2940gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata 3000tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt 3060ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga acaataaaac 3120tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt 3180cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 3240ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag 3300agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca 3360gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 3420ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag 3480aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 3540tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc 3600aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta 3660atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg 3720attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 3780taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 3840tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 3900atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt 3960tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg 4020acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt 4080cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 4140gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 4200taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 4260ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 4320tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 4380ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 4440cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 4500agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 4560gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 4620atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 4680gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 4740gctggccttt tgctcacatg t 4761 SEQ ID NO: 25aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 60(psPAX2atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 120plasmid)gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 180atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 240ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 300cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 360agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 420cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 480tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 540agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 600gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 660gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 720ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 780tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 840tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 900gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 960caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 1020atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggt 1080cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 1140ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 1200aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 1260actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat 1320caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 1380tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 1440ttagtcatcg ctattaccat gggtcgaggt gagccccacg ttctgcttca ctctccccat 1500ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 1560gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 1620gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 1680cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 1740gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc 1800ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 1860gctgtaatta gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc 1920ttaaagggct ccgggagggc cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg 1980tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg 2040ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg 2100tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg 2160ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt aacccccccc tgcaccgccc 2220tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc 2280ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 2340gcctcgggcc ggggagggct cgggggaggg gcgcggcggc cccggagcgc cggcggctgt 2400cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 2460cttcctttgt cccaaatctg gcggagccga aatctgggag gcgccgccgc accccctcta 2520gcgggcgcgg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg 2580tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgcc gcagggggac 2640ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc 2700tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac 2760gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgggccggc cgcgttgacg 2820cgcacggcaa gaggcgaggg gcggcgactg gtgagagatg ggtgcgagag cgtcagtatt 2880aagcggggga gaattagatc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa 2940atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc 3000tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct 3060tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt 3120gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca 3180aaacaaaagt aagaaaaaag cacagcaagc agcagctgac acaggacaca gcaatcaggt 3240cagccaaaat taccctatag tgcagaacat ccaggggcaa atggtacatc aggccatatc 3300acctagaact ttaaatgcat gggtaaaagt agtagaagag aaggctttca gcccagaagt 3360gatacccatg ttttcagcat tatcagaagg agccacccca caagatttaa acaccatgct 3420aaacacagtg gggggacatc aagcagccat gcaaatgtta aaagagacca tcaatgagga 3480agctgcagaa tgggatagag tgcatccagt gcatgcaggg cctattgcac caggccagat 3540gagagaacca aggggaagtg acatagcagg aactactagt acccttcagg aacaaatagg 3600atggatgaca cataatccac ctatcccagt aggagaaatc tataaaagat ggataatcct 3660gggattaaat aaaatagtaa gaatgtatag ccctaccagc attctggaca taagacaagg 3720accaaaggaa ccctttagag actatgtaga ccgattctat aaaactctaa gagccgagca 3780agcttcacaa gaggtaaaaa attggatgac agaaaccttg ttggtccaaa atgcgaaccc 3840agattgtaag actattttaa aagcattggg accaggagcg acactagaag aaatgatgac 3900agcatgtcag ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag 3960ccaagtaaca aatccagcta ccataatgat acagaaaggc aattttagga accaaagaaa 4020gactgttaag tgtttcaatt gtggcaaaga agggcacata gccaaaaatt gcagggcccc 4080taggaaaaag ggctgttgga aatgtggaaa ggaaggacac caaatgaaag attgtactga 4140gagacaggct aattttttag ggaagatctg gccttcccac aagggaaggc cagggaattt 4200tcttcagagc agaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga 4260gacaacaact ccctctcaga aggaggagcc gatagacaag gaactgtatc ctttagcttc 4320cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg gcaattaaag 4380gaagctctat tagatacagg agcagatgat acagtattag aagaaatgaa tttgccagga 4440agatggaaac caaaaatgat agggggaatt ggaggtttta tcaaagtagg acagtatgat 4500cagatactca tagaaatctg cggacataaa gctataggta cagtattagt aggacctaca 4560cctgtcaaca taattggaag aaatctgttg actcagattg gctgcacttt aaattttccc 4620attagtccta ttgagactgt accagtaaaa ttaaagccag gaatggatgg cccaaaagtt 4680aaacaatggc cattgacaga agaaaaaata aaagcattag tagaaatttg tacagaaatg 4740gaaaaggaag gaaaaatttc aaaaattggg cctgaaaatc catacaatac tccagtattt 4800gccataaaga aaaaagacag tactaaatgg agaaaattag tagatttcag agaacttaat 4860aagagaactc aagatttctg ggaagttcaa ttaggaatac cacatcctgc agggttaaaa 4920cagaaaaaat cagtaacagt actggatgtg ggcgatgcat atttttcagt tcccttagat 4980aaagacttca ggaagtatac tgcatttacc atacctagta taaacaatga gacaccaggg 5040attagatatc agtacaatgt gcttccacag ggatggaaag gatcaccagc aatattccag 5100tgtagcatga caaaaatctt agagcctttt agaaaacaaa atccagacat agtcatctat 5160caatacatgg atgatttgta tgtaggatct gacttagaaa tagggcagca tagaacaaaa 5220atagaggaac tgagacaaca tctgttgagg tggggattta ccacaccaga caaaaaacat 5280cagaaagaac ctccattcct ttggatgggt tatgaactcc atcctgataa atggacagta 5340cagcctatag tgctgccaga aaaggacagc tggactgtca atgacataca gaaattagtg 5400ggaaaattga attgggcaag tcagatttat gcagggatta aagtaaggca attatgtaaa 5460cttcttaggg gaaccaaagc actaacagaa gtagtaccac taacagaaga agcagagcta 5520gaactggcag aaaacaggga gattctaaaa gaaccggtac atggagtgta ttatgaccca 5580tcaaaagact taatagcaga aatacagaag caggggcaag gccaatggac atatcaaatt 5640tatcaagagc catttaaaaa tctgaaaaca ggaaaatatg caagaatgaa gggtgcccac 5700actaatgatg tgaaacaatt aacagaggca gtacaaaaaa tagccacaga aagcatagta 5760atatggggaa agactcctaa atttaaatta cccatacaaa aggaaacatg ggaagcatgg 5820tggacagagt attggcaagc cacctggatt cctgagtggg agtttgtcaa tacccctccc 5880ttagtgaagt tatggtacca gttagagaaa gaacccataa taggagcaga aactttctat 5940gtagatgggg cagccaatag ggaaactaaa ttaggaaaag caggatatgt aactgacaga 6000ggaagacaaa aagttgtccc cctaacggac acaacaaatc agaagactga gttacaagca 6060attcatctag ctttgcagga ttcgggatta gaagtaaaca tagtgacaga ctcacaatat 6120gcattgggaa tcattcaagc acaaccagat aagagtgaat cagagttagt cagtcaaata 6180atagagcagt taataaaaaa ggaaaaagtc tacctggcat gggtaccagc acacaaagga 6240attggaggaa atgaacaagt agatgggttg gtcagtgctg gaatcaggaa agtactattt 6300ttagatggaa tagataaggc ccaagaagaa catgagaaat atcacagtaa ttggagagca 6360atggctagtg attttaacct accacctgta gtagcaaaag aaatagtagc cagctgtgat 6420aaatgtcagc taaaagggga agccatgcat ggacaagtag actgtagccc aggaatatgg 6480cagctagatt gtacacattt agaaggaaaa gttatcttgg tagcagttca tgtagccagt 6540ggatatatag aagcagaagt aattccagca gagacagggc aagaaacagc atacttcctc 6600ttaaaattag caggaagatg gccagtaaaa acagtacata cagacaatgg cagcaatttc 6660accagtacta cagttaaggc cgcctgttgg tgggcgggga tcaagcagga atttggcatt 6720ccctacaatc cccaaagtca aggagtaata gaatctatga ataaagaatt aaagaaaatt 6780ataggacagg taagagatca ggctgaacat cttaagacag cagtacaaat ggcagtattc 6840atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 6900gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 6960aattttcggg tttattacag ggacaggaga gatccagttt ggaaaggacc agcaaagctc 7020ctctggaaag gtgaaggggc agtagtaata caagataata gtgacataaa agtagtgcca 7080agaagaaaag caaagatcat cagggattat ggaaaacaga tggcaggtga tgattgtgtg 7140gcaagtagac aggatgagga ttaacacatg gaattctgca acaactgctg tttatccatt 7200tcagaattgg gtgtcgacat agcagaatag gcgttactcg acagaggaga gcaagaaatg 7260gagccagtag atcctagact agagccctgg aagcatccag gaagtcagcc taaaactgct 7320tgtaccaatt gctattgtaa aaagtgttgc tttcattgcc aagtttgttt catgacaaaa 7380gccttaggca tctcctatgg caggaagaag cggagacagc gacgaagagc tcatcagaac 7440agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaat gcaacctata 7500atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata 7560gtaatcatag aatataggaa aatggccgct gatcttcaga cctggaggag gagatatgag 7620ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 7680agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 7740agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac 7800gctgacggta caggccagac aattattgtc tggtatagtg caggaggaga acaatttgct 7860gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct 7920ccaagcaaga atcctagctg tggaaagata cctaaaggat caacagctcc tagggatttg 7980gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa 8040taaatctctg gaacagatct ggaatcacac gacctggatg gagtgggaca gagaaattaa 8100caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa 8160tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac 8220aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag 8280aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc 8340gtttcagacc cacctcccaa tcccgagggg acccgacagg cccgaaggaa tagaagaaga 8400aggtggagag agagacagag acagatccat tcgattagtg aacggatcct tggcacttat 8460ctgggacgat ctgcggagcc tgtgcctctt cagctaccac cgcttgagag acttactctt 8520gattgtaacg aggattgtgg aacttctggg acgcaggggg tgggaagccc tcaaatattg 8580gtggaatctc ctacaatatt ggagtcagga gctaaagaat agtgctgtta gcttgctcaa 8640tgccacagcc atagcagtag ctgaggggac agatagggtt atagaagtag tacaaggagc 8700ttgtagagct attcgccaca tacctagaag aataagacag ggcttggaaa ggattttgct 8760ataagctcga aacaaccggt acctctagaa ctatagctag cagatctttt tccctctgcc 8820aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa 8880tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat 8940gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat 9000gccatatgct ggctgccatg aacaaaggtg gctataaaga ggtcatcagt atatgaaaca 9060gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt 9120tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt 9180actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt 9240atgaagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg 9300tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 9360aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 9420ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat 9480agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 9540gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 9600gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac 9660ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 9720aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 9780catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 9840ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 9900gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 9960caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 10020tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 10080gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 10140ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 10200cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 10260tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 10320tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 10380cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 10440agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 10500agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 10560gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 10620aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 10680ggattttggt catgagatta tca 10703 SEQ ID NO: 26gtcgacggat cgggagatca attccggcac ctgtcctacg agttgcatga taaagaagac 60(pCIGO-VSV.Gagtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt 120plasmid)gaaggctctc aagggcatcg gtcgatgcag gaaaaggaca agcagcgaaa attcacgccc 180ccttgggagg tggcggcata tgcaaaggat agcactccca ctctactact gggtatcata 240tgctgactgt atatgcatga ggatagcata tgctacccgg atacagatta ggatagcata 300tactacccag atatagatta ggatagcata tgctacccag atatagatta ggatagccta 360tgctacccag atataaatta ggatagcata tactacccag atatagatta ggatagcata 420tgctacccag atatagatta ggatagccta tgctacccag atatagatta ggatagcata 480tgctacccag atatagatta ggatagcata tgctatccag atatttgggt agtatatgct 540acccagatat aaattaggat agcatatact accctaatct ctattaggat agcatatgct 600acccggatac agattaggat agcatatact acccagatat agattaggat agcatatgct 660acccagatat agattaggat agcctatgct acccagatat aaattaggat agcatatact 720acccagatat agattaggat agcatatgct acccagatat agattaggat agcctatgct 780acccagatat agattaggat agcatatgct atccagatat ttgggtagta tatgctaccc 840atggcaacat tagcccaccg tgctctcagc gacctcgtga atatgaggac caacaaccct 900gtgcttggcg ctcaggcgca agtgtgtgta atttgtcctc cagatcgcag caatcgcgcc 960cctatcttgg cccgcccacc tacttatgca ggtattcccc ggggtgccat tagtggtttt 1020gtgggcaagt ggtttgaccg cagtggttag cggggttaca atcagccaag ttattacacc 1080cttattttac agtccaaaac cgcagggcgg cgtgtggggg ctgacgcgtg cccccactcc 1140acaatttcaa aaaaaagagt ggccacttgt ctttgtttat gggccccatt ggcgtggagc 1200cccgtttaat tttcgggggt gttagagaca accagtggag tccgctgctg tcggcgtcca 1260ctctctttcc ccttgttaca aatagagtgt aacaacatgg ttcacctgtc ttggtccctg 1320cctgggacac atcttaataa ccccagtatc atattgcact aggattatgt gttgcccata 1380gccataaatt cgtgtgagat ggacatccag tctttacggc ttgtccccac cccatggatt 1440tctattgtta aagatattca gaatgtttca ttcctacact agtatttatt gcccaagggg 1500tttgtgaggg ttatattggt gtcatagcac aatgccacca ctgaaccccc cgtccaaatt 1560ttattctggg ggcgtcacct gaaaccttgt tttcgagcac ctcacataca ccttactgtt 1620cacaactcag cagttattct attagctaaa cgaaggagaa tgaagaagca ggcgaagatt 1680caggagagtt cactgcccgc tccttgatct tcagccactg cccttgtgac taaaatggtt 1740cactaccctc gtggaatcct gaccccatgt aaataaaacc gtgacagctc atggggtggg 1800agatatcgct gttccttagg acccttttac taaccctaat tcgatagcat atgcttcccg 1860ttgggtaaca tatgctattg aattagggtt agtctggata gtatatacta ctacccggga 1920agcatatgct acccgtttag ggttaacaag ggggccttat aaacactatt gctaatgccc 1980tcttgagggt ccgcttatcg gtagctacac aggcccctct gattgacgtt ggtgtagcct 2040cccgtagtct tcctgggccc ctgggaggta catgtccccc agcattggtg taagagcttc 2100agccaagagt tacacataaa ggcaatgttg tgttgcagtc cacagactgc aaagtctgct 2160ccaggatgaa agccactcaa gggatcttca atattggcca ttagccatat tattcattgg 2220ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa 2280tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac 2340tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 2400cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 2460gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 2520atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 2580aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 2640catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 2700catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 2760atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 2820ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 2880acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatca ctagaagctt 2940tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt 3000ctcgaactta agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac 3060aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg 3120tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt 3180ccactcccag ttcaattaca gctcttaagg ctagagtact taatacgact cactataggc 3240tagcggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcaacagag 3300atcgatctgt ttccttgaca ctatgaagtg ccttttgtac ttagcctttt tattcattgg 3360ggtgaattgc aagttcacca tagtttttcc acacaaccaa aaaggaaact ggaaaaatgt 3420tccttctaat taccattatt gcccgtcaag ctcagattta aattggcata atgacttaat 3480aggcacagcc atacaagtca aaatgcccaa gagtcacaag gctattcaag cagacggttg 3540gatgtgtcat gcttccaaat gggtcactac ttgtgatttc cgctggtatg gaccgaagta 3600tataacacag tccatccgat ccttcactcc atctgtagaa caatgcaagg aaagcattga 3660acaaacgaaa caaggaactt ggctgaatcc aggcttccct cctcaaagtt gtggatatgc 3720aactgtgacg gatgccgaag cagtgattgt ccaggtgact cctcaccatg tgctggttga 3780tgaatacaca ggagaatggg ttgattcaca gttcatcaac ggaaaatgca gcaattacat 3840atgccccact gtccataact ctacaacctg gcattctgac tataaggtca aagggctatg 3900tgattctaac ctcatttcca tggacatcac cttcttctca gaggacggag agctatcatc 3960cctgggaaag gagggcacag ggttcagaag taactacttt gcttatgaaa ctggaggcaa 4020ggcctgcaaa atgcaatact gcaagcattg gggagtcaga ctcccatcag gtgtctggtt 4080cgagatggct gataaggatc tctttgctgc agccagattc cctgaatgcc cagaagggtc 4140aagtatctct gctccatctc agacctcagt ggatgtaagt ctaattcagg acgttgagag 4200gatcttggat tattccctct gccaagaaac ctggagcaaa atcagagcgg gtcttccaat 4260ctctccagtg gatctcagct atcttgctcc taaaaaccca ggaaccggtc ctgctttcac 4320cataatcaat ggtaccctaa aatactttga gaccagatac atcagagtcg atattgctgc 4380tccaatcctc tcaagaatgg tcggaatgat cagtggaact accacagaaa gggaactgtg 4440ggatgactgg gcaccatatg aagacgtgga aattggaccc aatggagttc tgaggaccag 4500ttcaggatat aagtttcctt tatacatgat tggacatggt atgttggact ccgatcttca 4560tcttagctca aaggctcagg tgttcgaaca tcctcacatt caagacgctg cttcgcaact 4620tcctgatgat gagagtttat tttttggtga tactgggcta tccaaaaatc caatcgagct 4680tgtagaaggt tggttcagta gttggaaaag ctctattgcc tcttttttct ttatcatagg 4740gttaatcatt ggactattct tggttctccg agttggtatc catctttgca ttaaattaaa 4800gcacaccaag aaaagacaga tttatacaga catagagatg aaccgacttg gaaagtaact 4860caaatcctgc acaacagatt cttcatgttt ggaccaaatc aacttgtgat accatgctca 4920aagaggcctc aattatattt gagtttttaa tttttatgga attctgcaga tatccatcac 4980actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct 5040agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 5100tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 5160gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 5220caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 5280tctatggctt ctgaggcgga aagaaccagc tgcattaatg aatcggccaa cgcgcgggga 5340gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5400tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 5460aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 5520gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 5580aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 5640ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 5700tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 5760tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 5820ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 5880tatcgccact ggcaggagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 5940ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 6000tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6060aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6120aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6180aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 6240ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 6300acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 6360ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 6420gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 6480taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6540tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 6600gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 6660cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6720aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6780cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6840tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6900gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6960tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7020gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7080ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7140cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7200agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7260gggttccgcg cacatttccc cgaaaagtgc cacctgac 7298

Expression of CD86 and 4-1BBL on engineered MOLM-14 aAPCs (also referredto herein as aMOLM14 aAPCs) was confirmed using flow cytometry (Canto IIflow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA),with results shown in FIG. 12. aMOLM-14 aAPCs were γ-irradiated at 100Gy and frozen.

Example 4—Expansion of Tumor Infiltrating Lymphocytes Using MOLM-14Artificial Antigen Presenting Cells

Engineered MOLM-14 cells were gamma-irradiated at 100 Gy beforeco-culturing with TILs. REPs were initiated by culturing TILs withirradiated, engineered MOLM-14 cells at 1:100 ratios in CM2 mediacontaining OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) for 14 days. At REPharvest, the TIL expansion rates, phenotype for activation anddifferentiation stage markers, metabolism rate, cytotoxicity andre-rapid expansion protocol (re-REP) assay were measured.

The results are shown in FIG. 13, FIG. 14, FIG. 15, and FIG. 16, wheretwo expansions for two sets of patient TILs are compared. The resultswith the CD86/4-1BBL modified MOLM-14 cells (labeled “TIL+EngineeredMOLM14+OKT3”) are comparable to the PBMC feeders (labeled“TIL+Feeders+OKT3”).

The results at day 14 are compared in FIG. 17, where results from twoadditional patient TILs are shown. The results indicate that MOLM-14cells that were engineered with CD86 and 4-1BBL showed similar TILexpansion in the rapid expansion protocol when compared with allogeneicfeeder cells. However, TILs cultured with parental MOLM-14 did notexpand.

In addition, TILs expanded against MOLM-14 maintained a TIL phenotypeand showed potency to kill P815 cells as measured using BRLA, which isdescribed in detail in Example 9. Briefly, luciferin-transduced P815target cells and TILs of interest were co-cultured with and withoutanti-CD3 to determine whether tumor reactivity of TILs is through TCRactivation (specific killing) or non-specific killing. Following 4 hoursof incubation, luciferin was added to the wells and incubated for 5minutes. After the incubation, bioluminescence intensity was read usinga luminometer. The percentage cytotoxicity and percentage survival werecalculated using the following formula: % Survival=(experimentalsurvival-minimum)/(maximum signal-minimum signal)×100 or %Cytotoxicity=100−(% Survival).

In FIG. 18, the results of expansions performed with low ratios of TILsto MOLM-14 aAPCs are shown in comparison to the results of expansionswith PBMC feeders. TILs (2×10⁴) were cultured at different TIL to aAPCor PBMC ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”,respectively) with parental MOLM-14 (“MOLM14”) cells, MOLM-14 cellstransduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders(“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in a 24-wellG-Rex plate. A control was performed using only OKT-3 (30 ng/mL) andIL-2 (3000 IU/mL) (“PBMC-”). Each condition was cultured in triplicate.Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cellswere counted on Day 7. FIG. 18 shows the mean plus standard deviation(SD) of viable cell numbers counted on Day 11, with ap-value calculatedby the student t-test. Additional control experiments were performedusing TILs alone, PBMCs alone, and aMOLM-14 cells alone, all of whichresulted in undetectable cell numbers (data not shown). The results showthat a ratio of 1:100 (TIL:aMOLM14) with OKT-3 and IL-2 yields a similarexpansion when compared to PBMC feeders with OKT-3 and IL-2 (p=0.0598).

In FIG. 19, the results of expansions performed with higher ratios ofTILs to MOLM-14 aAPCs, and otherwise performed as described above forFIG. 18, are shown in comparison to the results of expansions with PBMCfeeders. At a ratio of 1:300, the CD86/4-1BBL modified MOLM-14 aAPCswith OKT-3 and IL-2 significantly outperform PBMC feeders with OKT-3 andIL-2. These results were verified using different TIL batches in repeatexperiments shown in FIG. 20 and FIG. 21. In particular, as seen in FIG.21, TIL to aMOLM14 ratios of 1:200 show enhanced TIL expansion comparedto PBMC feeders under the same conditions. These results confirm thataMOLM14 aAPCs are unexpectedly superior in terms of expanding the TILnumbers than PBMCs particularly when using TIL:aMOLM14 ratios of 1:200to 1:300.

In FIG. 22 and FIG. 23, TILs expanded with aMOLM14 or PBMC were comparedby flow cytometry analysis to confirm that the TILs exhibited a similarphenotype and would be expected to perform similarly upon reinfusioninto a patient. Briefly, TILs were first stained with L/D Aqua todetermine viability. Next, cells were surface stained with TCR α/βPE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28PE, CD27 APC-C7, andCD57-PerCP-Cy5.5. Phenotype analysis was done by gating 10,000 to100,000 cells according to forward light scattering (FSC)/side lightscattering (SSC) using a Canto II flow cytometer (Becton, Dickinson, andCo., Franklin Lakes, N.J., USA). Data was analyzed by Cytobank softwareto create sunburst diagrams and SPADE (Spanning Tree Progression ofDensity Normalized Event) analyses. Gates were set based on fluorescenceminus one (FMO) controls. TILs expanded against aMOLM14 increases CD8⁺TILs when compared to PBMC feeders. Without being bound by theory, thisenhanced CD8⁺ TIL percentage may be due to the presence of 4-1BBLengineered to MOLM14. There is no difference in the expression of CD28,CD57, and CD27 differentiation markers. Additional flow cytometry datais shown in FIG. 24, and depicts a flow cytometry contour plot showing amemory subset (CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4⁺ or CD8⁺TILs, indicating that the memory subset obtained with PBMC feeders isreplicated by the aMOLM14 aAPCs.

The CD4 and CD8 SPADE tree of TILs expanded with aMOLM14 aAPCs or PBMCfeeders using CD3+ cells is shown in FIG. 25 and FIG. 26. The colorgradient is proportional to the mean fluorescence intensity (MFI) ofLAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without beingbound by theory, the results show that two batches of TILs expandedagainst aMOLM14 had undergone activation, but there was no difference inMFI between the aMOLM14 aAPCs and PBMC feeders, indicating that theaMOLM14 aAPCs effectively replicate the TIL phenotypic results obtainedwith PBMC feeders.

TILs expanded against aMOLM14 or PBMC were also analyzed for metabolicprofiles. Oxygen consumption rate (OCR) and extracellular acidificationrate (ECAR) of TILs after expansion with irradiated PBMC feeders oraMOLM14 aAPCs were measured using a dual mitochondrial-glycolytic stresstest. Briefly, cells were washed in assay medium (XF Assay Medium,Agilent Technologies, Santa Clara, Calif., USA), supplemented with 10 mMglucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, at pH 7.4, and then1×10⁵ viable cells were plated onto an adhesive-coated (Cell-Tak™,Corning) XFp cell culture microplate. Plates were spun to adhere thecells to the plate, then equilibrated at 37° C. in a humidified, non-CO₂incubator prior to analysis of cellular metabolism. Mitochondrial andglycolytic stress test experiments were performed using a Seahorse XFpAnalyzer (Agilent Technologies, Santa Clara, Calif., USA), sequentiallyinjecting the following compounds at specified intervals forsimultaneous analysis of mitochondrial and glycolytic respiration of thecells: 1 μM oligomycin; 0.5 μM FCCP; 50 mM 2-deoxyglucose; and 0.5 μMeach of rotenone and antimycin A. Results were analyzed using WAVEv2.3.0 software (Agilent Technologies, Santa Clara, Calif., USA) andGraphPad Prism v6.07 graphing software and are shown in FIG. 27 and FIG.28, where points represent mean±SEM measured in triplicate. Both TILsgrown with aMOLM14 aAPCs and PBMC feeders show similar oxphos andglycolysis behavior. This data suggests that aMOLM14 does not alter themetabolic programming of TILs when compared with PBMC feeders.

Example 5—Preparation of EM-3 Artificial Antigen Presenting Cells (aEM3aAPCs)

EM-3 cells were obtained from Creative Bioarray, Inc. (Shirley, N.Y.,USA). To develop an EM-3 based artificial APC, EM-3 cell lines wereengineered with CD86, 4-1BBL, and antibody against IgG Fc region (Clone7C12 or Clone 8B3). Human CD86 and human 4-1BBL/CD137 genes were clonedinto commercially-available PLV430G and co-transfected with PDONR221vectors (Invitrogen) using a lentiviral transduction method. The gatewaycloning method was used as described in Katzen, Expert Opin. Drug Disc.2007, 4, 571-589, to clone hCD86 and hCD137L genes onto the PLV430G andPDONR221 vectors. The 293T cell line was used for lentiviral production,and transduced to EM-3 cell lines. The transfected cells were sorted(S3e Cell Sorter, BioRad, Hercules, Calif., USA) using APC-conjugatedCD86 and PE-conjugated CD137L to isolate and enrich the cells. Theenriched cells were checked for purity by flow cytometry. Single-chainFv (scFv) antibody clones designated 7C12 and 8B3 were generated againstFc of mouse IgG1, IgG2a and IgG2b (Viva Biotech Ltd., Chicago, Ill.,USA). The amino acid sequences of these scFv clones are given in Table 7(SEQ ID NO:27 and SEQ ID NO:28). The generated scFv clones were screenedfor Fc binding efficiency against OKT-3, engineered towards pLV4301Gcontaining eGFP as co-reporter to produce lentivirus. The 293T cell linewas used for packaging and lentiviral production. Engineered EM-3(CD86/CD137L) cells were transduced using the lentiviral system andsorted using eGFP. EM37C12CD86CD137L and EM38B3CD86CD137L were regularlyassessed for the consistent expression of each transduced molecule byflow cytometry.

TABLE 7 Amino acid sequences of scFv clones 7C12 and 8B3. Identifier(Description) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 27QVQLVQSGGG LVKPGGSLRL SCAASGFNFN DQYMSWIRQA PGKGLEWVSF ISGSGGTTYY 60(mFC-7C12TDSVKGRFTI SRDNTKDSLY LQMNSLTVED TAVYYCARGG NYYTSVGRGT LVTVSAGGGG 120scFv) SGAPDIQMTQ SPGTLSLSPG ERAILSCRAS QSVSGYLAWY QQKPGQAPRL LIYGASSRAT180 GIPDRFSGSG SGTDFTLTIS SLRPEDIGTY YCKQYINAPF TFGGGTKVEI K 231SEQ ID NO: 28QVQLQQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGW ISPYNGNTDY 60(mFC-8B3 scFv)AQKVQGRVTL TTDTSTSTAY MELRSLRSDD TAVYYCATGG GTWYSDLWGR GTLVTVSAGG 120GGSGGGGSGG GGSGAPEIVL TQSPSTLSAS VGDRVSITCR ASQSIGGSLA WYQQKPGKAP 180KLLISEASTL ERGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQKYNSV PLTFGPGTKV 240EIK 243

A non-limiting protocol for preparation of aEM3 aAPCs, which may also beadapted for use with aMOLM14 aAPCs, is described in the followingparagraphs.

Molecular cloning of plasmids of interest may be performed as follows.To generate DONR vector the following cocktail may be used: B siteflanked PCR product or destination vector (e.g., Gateway-adaptedlentivector) 50-100 μg; DONR vector (e.g., pDONR222) 50-100 μg; BRClonase II (Life Technologies) 1 μL; and TE buffer ((1 mM Tris, 0.1 mMEDTA, pH 8.0, q.s. to bring volume to 5 μL). Incubate at roomtemperature for at least 1 hour. After incubation perform bacterialtransformation either by heat shock method or electroporation. Togenerate destination vector, the following cocktail may be used:recombined pDONR vector (e.g., pDON222-geneX) 50-100 μg, destinationvector (e.g., Gateway adapted lentivector) 50-100 μg, LR Clonase II(Life Technologies) 1 μL, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH8.0, q.s. to bring volume to 5 μL). Incubate at room temperature for atleast 1 hour. After incubation, perform bacterial transformation eitherby chemical competent transformation/heat shock method.

Transformation and selection of the cloned plasmid may be performed asfollows. The chemical competent transformation method may be performedas follows. Prepare nutrient agar plates (LB-Lennox or YT) withantibiotic for selection. Ensure that Recovery Medium (supplied byLucigen, Middleton, Wis., USA) is readily available at room temperature.Optionally, sterile culture tubes may be chilled on ice (e.g., 17 mm×100mm tubes (14 mL tube)), one tube for each transformation reaction).Remove E. cloni cells (Lucigen) from an −80° C. freezer and thawcompletely on wet ice (5-15 minutes). Optionally add 40 μL of E. clonicells to the chilled culture tube. Add 1-4 μL of DNA sample to the 40 μLof cells. Flick with finger (do not pipet up and down to mix, which canintroduce air bubbles and warm the cells). Incubate the cell/DNA mixtureon ice for 30 minutes. Heat shock cells by placing the culture tubes ina 42° C. water bath for 45 seconds. Return the 1.7 mL tube or culturetubes to ice for 2 minutes. Add 350 μL room temperature Recovery Mediumto the cells or 960 μL of room temperature Recovery Medium to the cellsin the culture tube. Place the tubes in a shaking incubator at 250 rpmfor 1 hour at 37° C. Plate up to 100% of the transformation mixture onLB-Lennox or YT agar plates containing the appropriate antibiotic. Theplating volume may need to be optimized depending on DNA. Incubate theplates overnight at 37° C. Transformed clones can be further grown inany rich culture medium (e.g., LB or TB).

Colonies for Miniprep (Qiagen, Inc., Valencia, Calif., USA) may be grownas follows. After colonies have formed from plating recoveredtransformation reaction of DNA manipulation (e.g. LR reaction), add 1 mLdesired TB/antibiotics into desired number of 2 mL Eppendorf microtubeswith punctured caps. Pick desired number of colonies using ART LTS 20soft pipette tip (VWR 89031-352) or 10 μL Denville tip. Place tip in 2mL Eppendorf microtube with punctured cap. Cut the tip so that it fitsin tube, close cap, and place tubes on shaker (purple 15 mL tube holderwith VWR brand 15 mL tubes). Shake overnight (for no more than 16 hours)at 225 rpm/37° C. After overnight incubation, place each tip in a 1 mLtube in a ClavePak 96 plate from Denville with sterile water in it (tosave the tip for making bacterial stock production after the plasmidsare screened and selected). Perform Miniprep according to the QiagenMini prep kit protocol (Qiagen, Inc., Valencia, Calif., USA). Once theplasmids are eluted, restriction digestion is performed to select theright clones. After selecting the plasmids, use the tips saved from thesame plasmids clone to grow the E. coli with the plasmid to makebacterial stock.

Lentiviral production may be performed as follows. The following mediacomposition is prepared: 500 mL DMEM/F12 (Sigma); 25 mL FBS HeatInactivated (HI) (Hyclone); 10 mM HEPES (Life Technologies); 1× Primocin(Invivogen); 1× Plasmocin (Invivogen); and 1×2-mermactoethanol (LifeTechnologies). Harvest T75 flasks (Thermo Fisher Scientific) containing90% confluent 293T cells. Aspirate media. Add 10 ml PBS, rinse gentlyand aspirate off. Add 2 mL TrypLE Express (Life Technologies) and evenlydistribute it over the cell layer, let sit for 3-5 minutes at 37° C.(cell culture incubator). Add 10 mL media and disperse cells bypipetting up and down. Combine if there are multiple flasks. Countcells. If using a hemacytometer to determine concentration, cells/mL=(#counted cells×dilution factor×10⁴). To split back into T75 flasks,determine the time at which the cells will need to be fully confluentand dilute accordingly. (Cells double every 16-18 hours, so 3 days=1/27dilution). Generally, a multiplication factor of 2.5 per day may be usedwhere confluence is 2×10⁵ cells/cm². Bring volume up to 25 mL of media.To plate for titration of stocks, each well of the assay requires 5×10⁴cells in 0.4 mL of media. Adjust 293T cells to 2×10⁴/mL in media. Plate1 mL per well in a 24 well plate. For example, cells plated Monday maybe infected on Tuesday and run on the flow cytometer on Friday, andcells plated Thursday are infected Friday and run on the flow cytometeron Monday. To plate for packaging transfections, seed T75 flasks with6.8×10⁶ cells one day before transfection or 1.7×10⁶ cells on themorning of transfection. (Seeding on the day of transfection may reducethe variation in transfection efficiency). Bring volume in flask up to25 mL with media. For example, flasks set up Monday are transfectedTuesday, and virus is collected on Thursday and Friday. In some cases(e.g., high titering constructs), the second collection can be omitted.To package lentiviral vectors, each T75 flask transfection requires 2 μgBaculo p35 plasmid (optional; only necessary if packaging a death gene),2 μg VSV.G env plasmid (e.g., pMD2.G or PCIGO VSV-G); 4.7 μsGag/polymerase plasmid (e.g., psPAX2 or pCMV-deltaR8.91), and 2.3 μg ofthe lentiviral vector described above. Determine the amount of VSV andR8.2/9.1 (+/−Baculo) plasmids needed for all samples (make a mixture ofthese DNAs if preparing many samples). Each T75 transfection requires 90μL LipofectAmine 2000 (Thermo Fisher Scientific) in 2 mL Opti-MEM medium(Thermo Fisher Scientific). Make a mix containing enough Opti-Mem andLipofectAmine 2000 for all samples. Mix gently and let sit for 5 minutesat room temp, and label as tube A. For each transfection, add packagingDNA and specific lentiviral vector DNA to 500 μL room temperatureOpti-MEM medium to a microtube and mix, and label as tube B. Add the 500μL of DNA from tube B to the 2 mL of the LipofectAmine 2000 mix in tubeA and mix gently, and incubate for 20-30 minutes at room temperature.Aspirate media from packaging flasks. Add the 2.5 mL ofDNA/Lipofectamine complexes to 5 mL Opti-MEM medium and add to cells (donot pipet directly on cells since 293T cells are only semi adherent).Process plates in small groups to avoid drying. Incubate overnight andchange media the next day in the morning. Collect the supernatant after24 hours of media change. Supernatants can be harvested in a singlecollection, 48 hours after transfection or as 2 collections, 48 and 72hours after transfection (in which case, harvests are pooled). If doublecollection is desired, collect supernatants by pipet on the first day,and replace with 20 mL of fresh media. To avoid flasks drying, work withonly 5 flasks at a time. Keep collected supernatants at 4° C. untilpooling the next day. Cool supernatants again on the following day andpool as appropriate. Spin the supernatants at 2000 rpm for 5 minutes tosediment any contaminating 293T cells. Filter harvested supernatantsthrough a 0.45 μm or 0.8 μm filter unit containing a pre-filter disc.Use a large enough filtration unit so that the filtration speed isrelatively fast. Store at 4° C. until ready to concentrate.

Virus may be concentrated using the PEG-it method (System Biosciences,Inc., Palo Alto, Calif. 94303) for longer-term storage at −80° C.Collect the supernatant from the transfection plates. Spin down the celldebris in the supernatant. The supernatant may also be filtered tocompletely remove any packaging cells. Add an amount of PEG-it solutionequal to a quarter of the volume of supernatant to the supernatant.Incubate the suspension at 4° C. for overnight. Centrifuge at 3500 rpm(1500 g) at 4° C. for 30 minutes. Remove supernatant and centrifuge at3500 rpm at 4° C. for 5 minutes. Remove remaining supernatant. Resuspendvirus in desired amount of phosphate-buffered saline (PBS) and freezealiquots at −80° C.

Transduction of cell line using lentivirus may be performed as follows.Adjust cells to be transduced to either: 1×10⁶ suspension cells per wellin 24 well plate (1 well per transduction) or 50% confluence foradherent cells (1 well per transduction) in 24 well plate. For suspendedcells, adjust concentration of cells to 1×10⁷/mL and plate 100 μL perwell in 24 well plate (1 well per transduction). For adherent cells,plate to achieve 50% confluence on day of transduction based oncells/cm′ (e.g., for 293T cells, confluence=2×10⁵/cm′). Total volume oftransduction per well should be approximately 500 μL with 3-10 μg/mLPolybrene (Hexadimethrine bromide, Sigma-Aldrich Co., St. Louis, Mo.,USA). The amount of concentrated virus added will depend on the MOI(multiplicity of infection) desired. A typical MOI is 10:1 but this mayvary depending on cell type. The transfection well should contain 100 μLof standard media containing either 1×10⁶ suspension cells or 50%confluent cells. For a MOI of 10:1 (e.g., virus activity is 1×10⁸ IU/mLand the target is to infect 1×10⁶ cells, then 1×10⁷ virions or 100 μL ofvirus is needed). Add standard media to 500 μL. Add Polybrene to 3 μg/mL(primary cells) to 10 μg/mL (tumor cell lines). Spin plate(s) at 1800rpm for 1.5 to 2 hours at 30° C. Incubate plate(s) at 37° C./5% CO₂using a Tissue Culture incubator for 5 hours to overnight. Change media.After 72 hours of transduction, if enough cells are available, performflow cytometric analysis to test the transduction efficiency.

Sorting of aAPCs may be performed as follows. Culture the cells in themedia described above until the cell count reaches a minimum of 10-20million. Take 1×10⁶ cells for each condition and stain with theantibodies for the proteins transduced. Wash the cells and analyze byflow cytometry to test the stability of transduction. Once theexpression of protein of interest has been analyzed and confirmed,prepare the rest of the cells for sorting. Sort the cells in an S3sorter by gating on markers of interest. Culture the sorted cells usingthe media mentioned above. Before freezing the vial, test the stabilityof the protein expression of interest. Use Recovery cell cultureFreezing media (Invitrogen), to make the cell bank of the same cells.Cells may be banked after each transduction and sorting procedure.

Nucleotide sequence information for the 7C12 and 8B3 scFv clones (SEQ IDNO:29 and SEQ ID NO:30) and their lentiviral vectors are given in Table8. Sequences used for generation of the pLV4301G 7C12 scFv mIgG hCD8flag vector are provided as SED IQ NO:31 to SEQ ID NO:34 and aredepicted in FIG. 29 to FIG. 32. Sequences used for generation of thepLV4301G 8B3 scFv mIgG hCD8 flag vector are provided as SEQ ID NO:35 toSEQ ID NO:38 and are depicted in FIG. 33 to FIG. 36.

TABLE 8Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.Identifier (Description) Sequence SEQ ID NO: 29caggtgcagc tggtgcagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60(mFC-7C12tcctgtgcag cctctggatt caatttcaat gaccagtaca tgagttggat ccgccaggct 120scFv) ccagggaagg ggctggagtg ggtttcattc attagtggta gtggtggtac cacatactac180 acagactctg tgaagggccg gttcaccatc tccagggaca acaccaagga ctcattgtat240 ttgcaaatga acagcctgac agtcgaggac acggccgtgt actactgtgc gagaggaggg300 aattattata cttcggtggg ccggggcacc ctggtcaccg tctcggccgg tggcggcgga360 tctggcgcgc cagacatcca gatgacccag tctccaggca ccctgtcttt gtctccaggg420 gaaagagcca tcctctcctg cagggccagt cagagtgtta gcggctacct agcctggtat480 caacagaaac ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact540 ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc600 agcctgcggc ctgaagatat tggaacatat tactgtaaac agtacattaa tgccccattc660 actttcggcg gcgggaccaa ggtggagatc aaa 693 SEQ ID NO: 30caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60(mFC-8B3 scFv)tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagccctt acaatggtaa cacagattat 180gcacagaagg tccagggcag agtcaccttg accacagaca catccacgag cacagcctac 240atggagctga ggagcctgag atccgacgac acggccgtgt attactgtgc gacaggtggc 300gggacctggt actccgatct ctggggccgt ggcaccctgg tcaccgtctc ggccggtggc 360ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgccaga aattgtgctg 420actcagtctc cctccaccct gtctgcatct gtaggagaca gagtcagcat cacttgccgg 480gccagtcaga gtattggtgg gtcgttggcc tggtatcaac aaaagccagg gaaagcccct 540aagctcctga tctctgaggc gtctacttta gagaggggcg tcccatcaag attcagcggc 600agtggatctg ggacagattt cactctcacc atcaggagcc tgcagcctga agatgttgca 660acttattact gtcaaaaata taacagtgtc ccgctcactt tcggccctgg gaccaaggtg 720gagatcaaa 729 SEQ ID NO: 31cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(destinationgttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120vector cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat180 pLV4301G)taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840aaggccatcc agcctcgcgt cg 9862 SEQ ID NO: 32ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60(donor vectorattttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 1201, pMK 7c12gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180anti mFC scFVgggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240CoOp ECORVgctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300SacII L1R5)acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc 480atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag 540gtgcagctgg tgcagtctgg cggcggactc gtgaaacctg gcggcagcct gagactgagc 600tgtgccgcca gcggcttcaa cttcaacgac cagtacatga gctggatccg gcaggcccct 660ggcaagggac tggaatgggt gtccttcatc agcggcagcg gcggcaccac ctactacacc 720gatagcgtga agggccggtt caccatcagc cgggacaaca ccaaggacag cctgtacctg 780cagatgaaca gcctgaccgt ggaagatacc gccgtgtact actgcgccag aggcggcaat 840tactacacca gcgtgggcag aggcaccctc gtgacagtgt ctgctggcgg aggcggatca 900ggcggcggag gatcaggggg aggcggaagc ggagcacccg atatccagat gacacagagc 960cccggcaccc tgtctctgag ccctggcgaa agagccatcc tgagctgcag agccagccag 1020agcgtgtccg gatacctggc ttggtatcag cagaagcccg gccaggcccc cagactgctg 1080atctatggcg ccaggaggag agccacaggc atccccgata gattcagcgg ctctggcagc 1140ggcaccgact tcaccctgac aatcagctcc ctgcggcccg aggacatcgg cacctactat 1200tgcaagcagt acatcaacgc ccccttcacc ttcggcggag gcaccaaggt ggaaatcaag 1260ccgcgggcca actttgtata caaaagtgga acgagaaacg taaaatgata taaatatcaa 1320tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat 1380ccagtcacta tgaatcaact acttagatgg tattagtgac ctgtactggg cctcatgggc 1440cttcctttca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaacatgg 1500tcatagctgt ttccttgcgt attgggcgct ctccgcttcc tcgctcactg actcgctgcg 1560ctcggtcgtt cgggtaaagc ctggggtgcc taatgagcaa aaggccagca aaaggccagg 1620aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgccgccc tgacgagcat 1680cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 1740gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 1800tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 1860tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accgcccgtt 1920cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 1980gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2040ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2100ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2160ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2220agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2280aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2340atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2400tctgacagtt attagaaaaa ttcatccagc agacgataaa acgcaatacg ctggctatcc 2460ggtgccgcaa tgccatacag caccagaaaa cgatccgccc attcgccgcc cagttcttcc 2520gcaatatcac gggtggccag cgcaatatcc tgataacgat ccgccacgcc cagacggccg 2580caatcaataa agccgctaaa acggccattt tccaccataa tgttcggcag gcacgcatca 2640ccatgggtca ccaccagatc ttcgccatcc ggcatgctcg ctttcagacg cgcaaacagc 2700tctgccggtg ccaggccctg atgttcttca tccagatcat cctgatccac caggcccgct 2760tccatacggg tacgcgcacg ttcaatacga tgtttcgcct gatgatcaaa cggacaggtc 2820gccgggtcca gggtatgcag acgacgcatg gcatccgcca taatgctcac tttttctgcc 2880ggcgccagat ggctagacag cagatcctga cccggcactt cgcccagcag cagccaatca 2940cggcccgctt cggtcaccac atccagcacc gccgcacacg gaacaccggt ggtggccagc 3000cagctcagac gcgccgcttc atcctgcagc tcgttcagcg caccgctcag atcggttttc 3060acaaacagca ccggacgacc ctgcgcgctc agacgaaaca ccgccgcatc agagcagcca 3120atggtctgct gcgcccaatc atagccaaac agacgttcca cccacgctgc cgggctaccc 3180gcatgcaggc catcctgttc aatcatactc ttcctttttc aatattattg aagcatttat 3240cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 3300ggggttccgc gcacatttcc ccgaaaagtg ccac 3334 SEQ ID NO: 33ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60(donor vectorattttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 1202, pMK hCD8agatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180scaffold TN L5gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg420 atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa480 cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga540 ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg600 cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc660 tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg720 gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt780 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg840 tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc900 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct960 tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt1020 aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc1080 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc1140 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga1200 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt1260 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg1320 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc1380 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg1440 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc1500 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg1560 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc1620 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct1680 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt1740 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa1800 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag1860 aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca1920 tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg1980 gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg2040 ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc2100 agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg2160 ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc2220 gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta2280 tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta2340 gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc2400 accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc2460 gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga2520 cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc2580 caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc2640 tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc2700 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca2760 tttccccgaa aagtgccac 2779 SEQ ID NO: 34cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(Final vectorgttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120used forcggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180lentiviraltaggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240production,ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300pLV4301G 7C12cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360scFV mIgG hCD8tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat2700 caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg2760 gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctggt gcagtctggc2820 ggcggactcg tgaaacctgg cggcagcctg agactgagct gtgccgccag cggcttcaac2880 ttcaacgacc agtacatgag ctggatccgg caggcccctg gcaagggact ggaatgggtg2940 tccttcatca gcggcagcgg cggcaccacc tactacaccg atagcgtgaa gggccggttc3000 accatcagcc gggacaacac caaggacagc ctgtacctgc agatgaacag cctgaccgtg3060 gaagataccg ccgtgtacta ctgcgccaga ggcggcaatt actacaccag cgtgggcaga3120 ggcaccctcg tgacagtgtc tgctggcgga ggcggatcag gcggcggagg atcaggggga3180 ggcggaagcg gagcacccga tatccagatg acacagagcc ccggcaccct gtctctgagc3240 cctggcgaaa gagccatcct gagctgcaga gccagccaga gcgtgtccgg atacctggct3300 tggtatcagc agaagcccgg ccaggccccc agactgctga tctatggcgc cagcagcaga3360 gccacaggca tccccgatag attcagcggc tctggcagcg gcaccgactt caccctgaca3420 atcagctccc tgcggcccga ggacatcggc acctactatt gcaagcagta catcaacgcc3480 cccttcacct tcggcggagg caccaaggtg gaaatcaagc cgcgggccaa ctttgtatac3540 aaaagtggcc cgcggacaac aacccctgcc cccagacctc ctaccccagc ccctacaatt3600 gccagccagc ctctgagcct gaggcccgag gcttgtagac ctgctgctgg cggagccgtg3660 cacaccagag gactggattt cgcctgcgac atctacatct gggcccctct ggccggcaca3720 tgtggcgtgc tgctgctgag cctcgtgatc accctgtact gcggctccac cagcggctcc3780 ggcaagcccg gctctggcga gggctccacc agcggcgact acaaggacga cgatgacaag3840 taataggata tcggttcagc tttcttgtac aaagttggga ttcgagttaa ttaagttaac3900 gaattccccc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa3960 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg4020 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc4080 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct4140 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaaccgccc acctggcgac4200 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc4260 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta4320 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg4380 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga4440 accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggga4500 ggcggaagcg gcggaggctc ccctcgaggc accatggtga gcaagggcga ggagctgttc4560 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc4620 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc4680 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg4740 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg4800 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc4860 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc4920 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac4980 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc5040 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc5100 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc5160 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg5220 atcactctcg gcatggacga gctgtacaag taacgcgtcc cgggtctaga gctagcggta5280 ccatgcatta cgtagtcgac gacttaatta agctagccta gtgccatttg ttcagtggtt5340 cgtagggctt tcccccactg tttggctttc agttatatgg atgatgtggt attgggggcc5400 aagtctgtac agcatcttga gtcccttttt accgctgtta ccaattttct tttgtctttg5460 ggtatacatt taaaccctaa caaaacaaag agatggggtt actctctaaa ttttatgggt5520 tatgtcattg gatgttatgg gtccttgcca caagaacaca tcatacaaaa aatcaaagaa5580 tgttttagaa aacttcctat taacaggcct attgattgga aagtatgtca acgaattgtg5640 ggtcttttgg gttttgctgc cccttttaca caatgtggtt atcctgcgtt gatgcctttg5700 tatgcatgta ttcaatctaa gcaggctttc actttctcgc caacttacaa ggcctttctg5760 tgtaaacaat acctgaacct ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg5820 tttgctgacg caacccccac tggctggggc ttggtcatgg gccatcagcg catgcgtgga5880 accttttcgg ctcctctgcc gatccatact gcggaactcc tagccgcttg ttttgctcgc5940 agcaggtctg gagcaaacat tatcgggact gataactctg ttgtcctatc ccgcaaatat6000 acatcgtttc catggctgct aggctgtgct gccaactgga tcctgcgcgg gacgtccttt6060 gtttacgtcc cgtcggcgct gaatcctgcg gacgaccctt ctcggggtcg cttgggactc6120 tctcgtcccc ttctccgtct gccgttccga ccgaccacgg ggcgcacctc tctttacgcg6180 gactccccgt ctgtgccttc tcatctgccg gaccgtgtgc acttcgcttc acctctgcac6240 gtcgcatgga gaccaccgtg aacgcccacc aaatattgcc caaggtctta cataagagga6300 ctcttggact ctcagcaatg tcaacgaccg accttgaggc atacttcaaa gactgtttgt6360 ttaaagactg ggaggagttg ggggaggaga ttaggttaaa ggtctttgta ctaggaggct6420 gtaggcataa attggtctgc gcaccagcac catggcgcaa tcactagagc ggggtacctt6480 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg6540 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt6600 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc6660 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg6720 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta6780 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga6840 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa6900 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca6960 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct7020 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc7080 agaggccgag gccggatccc ttgagtggct ttcatcctgg agcagacttt gcagtctgtg7140 gactgcaaca caacattgcc tttatgtgta actcttggct gaagctctta caccaatgct7200 gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc7260 agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt7320 ataaggcccc cttgttaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata7380 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt7440 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag7500 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca7560 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc7620 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat7680 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc7740 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg7800 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc7860 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat7920 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga7980 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc8040 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc8100 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt8160 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc8220 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc8280 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca8340 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca8400 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt8460 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta8520 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg8580 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc8640 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag8700 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa8760 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc8820 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc8880 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta8940 caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag9000 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct9060 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga9120 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc9180 ggccttttta cggttcctgg ccttttgctg gcctttttga agctgtccct gatggtcgtc9240 atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag9300 aagaatcata atggggaagg ccatccagcc tcgcgtcg 9338 SEQ ID NO: 35cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(destinationgttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120vector,cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180pLV4301G)taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840aaggccatcc agcctcgcgt cg 9862 SEQ ID NO: 36ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60(donor vectorattttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 1201, pMK 8B3gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180anti mFC scFVgggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240CoOp ECORVgctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300SacII L1R5)acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc 480atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag 540gtgcagctgc agcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 600tgcaaggcta gcggcggcac cttcaggagc tacgccattt cttgggtgcg ccaggcccct 660ggacagggcc tggaatggat gggctggatc agcccctaca acggcaacac cgactacgcc 720cagaaagtgc agggcagagt gaccctgacc accgacacca gcacctccac cgcctacatg 780gaactgcgga gcctgagaag cgacgacacc gccgtgtact actgtgccac aggcggcgga 840acctggtaca gcgatctgtg gggcagaggc accctcgtga cagtgtctgc tggcggcgga 900ggatctggcg gaggcggaag tggcggggga ggaagcggag cacctgagat cgtgctgacc 960cagagcccta gcacactgag cgccagcgtg ggcgacagag tgtccatcac ctgtagagcc 1020agccagagca tcggaggcag cctggcctgg tatcagcaga agcctggcaa ggcccccaag 1080ctgctgatct ctgaggccag caccctggaa agaggcgtgc ccagcagatt ttccggcagc 1140ggctctggca ccgacttcac cctgacaatc agcagcctgc agcccgagga cgtggccacc 1200tactactgcc agaagtacaa cagcgtgccc ctgaccttcg gccctggcac caaggtggaa 1260atcaagccgc gggccaactt tgtatacaaa agtggaacga gaaacgtaaa atgatataaa 1320tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca 1380acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt actgggcctc 1440atgggccttc ctttcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 1500acatggtcat agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc 1560gctgcgctcg gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag 1620gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccgccctgac 1680gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1740taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1800accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 1860tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1920cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1980agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2040gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 2100gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2160tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2220acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2280cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2340acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2400acttggtctg acagttatta gaaaaattca tccagcagac gataaaacgc aatacgctgg 2460ctatccggtg ccgcaatgcc atacagcacc agaaaacgat ccgcccattc gccgcccagt 2520tcttccgcaa tatcacgggt ggccagcgca atatcctgat aacgatccgc cacgcccaga 2580cggccgcaat caataaagcc gctaaaacgg ccattttcca ccataatgtt cggcaggcac 2640gcatcaccat gggtcaccac cagatcttcg ccatccggca tgctcgcttt cagacgcgca 2700aacagctctg ccggtgccag gccctgatgt tcttcatcca gatcatcctg atccaccagg 2760cccgcttcca tacgggtacg cgcacgttca atacgatgtt tcgcctgatg atcaaacgga 2820caggtcgccg ggtccagggt atgcagacga cgcatggcat ccgccataat gctcactttt 2880tctgccggcg ccagatggct agacagcaga tcctgacccg gcacttcgcc cagcagcagc 2940caatcacggc ccgcttcggt caccacatcc agcaccgccg cacacggaac accggtggtg 3000gccagccagc tcagacgcgc cgcttcatcc tgcagctcgt tcagcgcacc gctcagatcg 3060gttttcacaa acagcaccgg acgaccctgc gcgctcagac gaaacaccgc cgcatcagag 3120cagccaatgg tctgctgcgc ccaatcatag ccaaacagac gttccaccca cgctgccggg 3180ctacccgcat gcaggccatc ctgttcaatc atactcttcc tttttcaata ttattgaagc 3240atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3300caaatagggg ttccgcgcac atttccccga aaagtgccac 3340 SEQ ID NO: 37ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60(donor vectorattttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 1202, pMK hCD8agatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180scaffold TN L5gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg300 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca360 aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg420 atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa480 cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga540 ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg600 cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc660 tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg720 gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt780 tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg840 tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc900 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct960 tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt1020 aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc1080 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc1140 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga1200 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt1260 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg1320 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc1380 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg1440 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc1500 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg1560 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc1620 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct1680 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt1740 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa1800 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag1860 aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca1920 tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg1980 gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg2040 ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc2100 agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg2160 ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc2220 gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta2280 tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta2340 gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc2400 accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc2460 gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga2520 cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc2580 caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc2640 tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc2700 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca2760 tttccccgaa aagtgccac 2779 SEQ ID NO: 38cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60(Final vectorgttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120used forcggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180lentiviraltaggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240production,ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300pLV4301G 8B3cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360scFV mIgG hCD8tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac480 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat540 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg600 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc660 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct720 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg780 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg840 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag900 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat960 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag1020 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca1080 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata1140 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga1200 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc1260 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata1320 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag1380 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag1440 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat1500 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc1560 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa1620 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca1680 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc1740 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct1800 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata1860 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat1920 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag1980 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga2040 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat2100 ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg2160 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa2220 ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa2280 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg2340 caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag2400 agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag2460 ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc2520 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca2580 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc2640 cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat2700 caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg2760 gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctgca gcagtctggc2820 gccgaagtga agaaacccgg cagcagcgtg aaggtgtcct gcaaggctag cggcggcacc2880 ttcaggagct acgccatttc ttgggtgcgc caggcccctg gacagggcct ggaatggatg2940 ggctggatca gcccctacaa cggcaacacc gactacgccc agaaagtgca gggcagagtg3000 accctgacca ccgacaccag cacctccacc gcctacatgg aactgcggag cctgagaagc3060 gacgacaccg ccgtgtacta ctgtgccaca ggcggcggaa cctggtacag cgatctgtgg3120 ggcagaggca ccctcgtgac agtgtctgct ggcggcggag gatctggcgg aggcggaagt3180 ggcgggggag gaagcggagc acctgagatc gtgctgaccc agagccctag cacactgagc3240 gccagcgtgg gcgacagagt gtccatcacc tgtagagcca gccagagcat cggaggcagc3300 ctggcctggt atcagcagaa gcctggcaag gcccccaagc tgctgatctc tgaggccagc3360 accctggaaa gaggcgtgcc cagcagattt tccggcagcg gctctggcac cgacttcacc3420 ctgacaatca gcagcctgca gcccgaggac gtggccacct actactgcca gaagtacaac3480 agcgtgcccc tgaccttcgg ccctggcacc aaggtggaaa tcaagccgcg ggccaacttt3540 gtatacaaaa gtggcccgcg gacaacaacc cctgccccca gacctcctac cccagcccct3600 acaattgcca gccagcctct gagcctgagg cccgaggctt gtagacctgc tgctggcgga3660 gccgtgcaca ccagaggact ggatttcgcc tgcgacatct acatctgggc ccctctggcc3720 ggcacatgtg gcgtgctgct gctgagcctc gtgatcaccc tgtactgcgg ctccaccagc3780 ggctccggca agcccggctc tggcgagggc tccaccagcg gcgactacaa ggacgacgat3840 gacaagtaat aggatatcgg ttcagctttc ttgtacaaag ttgggattcg agttaattaa3900 gttaacgaat tccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg3960 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc4020 aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc4080 cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa4140 gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct4200 ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca4260 caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca4320 agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat4380 ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc4440 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc4500 atgggaggcg gaagcggcgg aggctcccct cgaggcacca tggtgagcaa gggcgaggag4560 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag4620 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc4680 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac4740 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc4800 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac4860 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag4920 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac4980 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag5040 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc5100 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc5160 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc5220 gccgggatca ctctcggcat ggacgagctg tacaagtaac gcgtcccggg tctagagcta5280 gcggtaccat gcattacgta gtcgacgact taattaagct agcctagtgc catttgttca5340 gtggttcgta gggctttccc ccactgtttg gctttcagtt atatggatga tgtggtattg5400 ggggccaagt ctgtacagca tcttgagtcc ctttttaccg ctgttaccaa ttttcttttg5460 tctttgggta tacatttaaa ccctaacaaa acaaagagat ggggttactc tctaaatttt5520 atgggttatg tcattggatg ttatgggtcc ttgccacaag aacacatcat acaaaaaatc5580 aaagaatgtt ttagaaaact tcctattaac aggcctattg attggaaagt atgtcaacga5640 attgtgggtc ttttgggttt tgctgcccct tttacacaat gtggttatcc tgcgttgatg5700 cctttgtatg catgtattca atctaagcag gctttcactt tctcgccaac ttacaaggcc5760 tttctgtgta aacaatacct gaacctttac cccgttgccc ggcaacggcc aggtctgtgc5820 caagtgtttg ctgacgcaac ccccactggc tggggcttgg tcatgggcca tcagcgcatg5880 cgtggaacct tttcggctcc tctgccgatc catactgcgg aactcctagc cgcttgtttt5940 gctcgcagca ggtctggagc aaacattatc gggactgata actctgttgt cctatcccgc6000 aaatatacat cgtttccatg gctgctaggc tgtgctgcca actggatcct gcgcgggacg6060 tcctttgttt acgtcccgtc ggcgctgaat cctgcggacg acccttctcg gggtcgcttg6120 ggactctctc gtccccttct ccgtctgccg ttccgaccga ccacggggcg cacctctctt6180 tacgcggact ccccgtctgt gccttctcat ctgccggacc gtgtgcactt cgcttcacct6240 ctgcacgtcg catggagacc accgtgaacg cccaccaaat attgcccaag gtcttacata6300 agaggactct tggactctca gcaatgtcaa cgaccgacct tgaggcatac ttcaaagact6360 gtttgtttaa agactgggag gagttggggg aggagattag gttaaaggtc tttgtactag6420 gaggctgtag gcataaattg gtctgcgcac cagcaccatg gcgcaatcac tagagcgggg6480 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa6540 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta6600 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc6660 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt6720 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta6780 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat6840 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca6900 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac6960 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc7020 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat7080 ttatgcagag gccgaggccg gatcccttga gtggctttca tcctggagca gactttgcag7140 tctgtggact gcaacacaac attgccttta tgtgtaactc ttggctgaag ctcttacacc7200 aatgctgggg gacatgtacc tcccaggggc ccaggaagac tacgggaggc tacaccaacg7260 tcaatcagag gggcctgtgt agctaccgat aagcggaccc tcaagagggc attagcaata7320 gtgtttataa ggcccccttg ttaattcttg aagacgaaag ggcctcgtga tacgcctatt7380 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg7440 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct7500 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat7560 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc7620 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg7680 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg7740 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga7800 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta7860 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc7920 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc7980 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg8040 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc8100 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca8160 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct8220 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat8280 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg8340 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat8400 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact8460 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat8520 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc8580 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct8640 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg8700 cttcaggaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca8760 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc8820 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga8880 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac8940 gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga9000 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag9060 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg9120 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag9180 caacgcggcc tttttacggt tcctggcctt ttgctggcct ttttgaagct gtccctgatg9240 gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga9300 agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcg 9344 SEQ ID NO: 39gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60(pLenti-C-Myc-atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120DDK OX40L)gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca 240ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 840tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 900agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 960ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1260attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1320gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2160ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt 3300accgaggaga tctgccgccg cgatcgccat ggaaagggtc caacccctgg aagagaatgt 3360gggaaatgca gccaggccaa gattcgagag gaacaagcta ttgctggtgg cctctgtaat 3420tcagggactg gggctgctcc tgtgcttcac ctacatctgc ctgcacttct ctgctcttca 3480ggtatcacat cggtatcctc gaattcaaag tatcaaagta caatttaccg aatataagaa 3540ggagaaaggt ttcatcctca cttcccaaaa ggaggatgaa atcatgaagg tgcagaacaa 3600ctcagtcatc atcaactgtg atgggtttta tctcatctcc ctgaagggct acttctccca 3660ggaagtcaac attagccttc attaccagaa ggatgaggag cccctcttcc aactgaagaa 3720ggtcaggtct gtcaactcct tgatggtggc ctctctgact tacaaagaca aagtctactt 3780gaatgtgacc actgacaata cctccctgga tgacttccat gtgaatggcg gagaactgat 3840tcttatccat caaaatcctg gtgaattctg tgtccttacg cgtacgcggc cgctcgagca 3900gaaactcatc tcagaagagg atctggcagc aaatgatatc ctggattaca aggatgacga 3960cgataaggtt taaacggccg gccgcggtct gtacaagtag gattcgtcga gggacctaat 4020aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg gttccactag 4080gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga 4140ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 4200cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 4260tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 4320actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 4380tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 4440gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 4500aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 4560tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 4620ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 4680tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc 4740aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga 4800ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa 4860ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 4920ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc 4980tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg 5040ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac 5100ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga 5160gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg 5220tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 5280cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 5340gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 5400tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5460ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5520gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5580cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5640gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5700tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5760cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5820cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5880gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5940agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6000cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6060tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6120tttggtcatg attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc 6180attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc 6240agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg 6300tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg 6360aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc 6420acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc 6480gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat 6540atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg 6600gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa 6660aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat 6720gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt 6780tttttctcca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6840atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cccgcgcaca 6900tttccccgaa aagtgccacc tgac 6924

In the preparations of engineered EM-3 aAPCs (also referred to herein asaEM3 aAPCs) used for the experiments described herein, expression ofCD86 and 4-1BBL was confirmed using flow cytometry (Canto II flowcytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), withresults shown in FIG. 37. aEM3 aAPCs were γ-irradiated at 100 Gy andfrozen.

aEM-3 cells previously transduced to express CD86, antibody against IgGFc region, and 4-1BBL (or optionally without 4-1BBL), as describedabove, were genetically engineered with a co-stimulatory human OX-40Lusing a similar lentiviral transduction approach. To generate lentiviruscontaining human OX-40L, pLenti-C-Myc-DDK OX40L (PS100064, Origene, SEQID NO:39, FIG. 90) vector together with the VSV-G envelope plasmid(pCIGO-VSV.G) were co-transfected into a Phoenix-GP (ATCC CRL-3215) cellline using PolyJet (Signagen Laboratories, Rockville, Md., USA). Thesupernatants were harvested 60 hours later and concentrated using AmiconUltra-15 Centrifugal Filter Unit with Ultracel-100 membrane. aEM-3 cellswere then infected with concentrated lentivirus and further expanded forfive days. The cells were stained with PE-conjugated anti-human OX40L,Brilliant Violet 421-conjugated anti-human CD137L (if 4-1BBL is includedin the prior aEM-3 cells), and PE/Cy7 conjugated anti-human CD86 andsorted based on the expression of GFP, OX40L, CD137L (when included),and CD86 using a S3e Cell Sorter (Bio-Rad, Inc., Hercules, Calif., USA).The purity of sorted cells was further validated using flow cytometry.The enriched cells were checked for purity by flow cytometry.

Example 6—Expansion of Tumor Infiltrating Lymphocytes Using EM-3Artificial Antigen Presenting Cells

Experiments were performed to test the ability of EM-3 aAPCs (aEM3) toexpand TILs. TIL were co-cultured with aEM3 (7C12 or 8B3) at a ratio of1:100 ratio plus OKT-3 (30 mg/mL) and IL-2 (3000 IU/mL). Cells werecounted on Day 11 and 14. The results are plotted for two batches ofTILs in FIG. 38 and FIG. 39. In addition, TILs were co-cultured withaEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL) with orwithout OKT-3 (30 mg/mL). The results are plotted in FIG. 40, where thebar graph shows cell numbers determined on Day 11.

FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs (aEM3)at different TIL:aAPC ratios. The results show that aEM3 aAPCs performcomparably to and in some cases better than PBMCs, particularly atratios of 1:200 at longer culture times (14 days).

FIG. 42 illustrates the low variability in cell counts from TILexpansions with EM-3 aAPCs (aEM3) in comparison to PBMC feeders. TILs(2×10⁴) were co-cultured with five different PBMC feeder lots or aEM3(in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 wellplate. The graph shows viable cell numbers (mean) with 95% confidenceinterval counted on Day 14. FIG. 43 compares the results of TILexpansions with EM-3 aAPCs and MOLM-14 aAPCs, to illustrate variabilityin cell counts for both aEM3 and aMOLM14 in comparison to TILs (2×10⁴)were co-cultured with five different PBMC feeder lots or aMOLM14 (intriplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000IU/mL) in a G-Rex 24 well plate. Viable cells were counted on day 14,and the graph shows viable cell numbers (mean) with 95% confidenceinterval. The aEM3 and aMOLM14 results indicate that much greaterconsistency can be obtained with both aAPCs compared to the PBMC feederapproach preferred in the prior art.

TILs expanded against aEM3 or PBMC feeders were used for flow cytometryanalysis using 4 different panels (differentiation panels 1 and 2, Tcell activation panels 1 and 2). Briefly, TILs were first stained withL/D Aqua to determine viability. Next, cells were surface stained withTCR α/β PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28 PE, CD27 APC-Cy7, andCD57-PerCP-Cy5.5 for differentiation panel 1; CD45RA PE-Cy7, CD8aPerCP/Cy5, CCR7 PE, CD4 FITC, CD3 APC-Cy7, CD38 APC, and HLA-DR PB, fordifferentiation panel 2; CD137 PE-Cy7, CD8a PerCP-Cy5.5, Lag3 PE, CD4FITC, CD3 APC-Cy7, PD1 APC, and Tim-3 BV421 for T cell activation panel1; or CD69 PE-Cy7, CD8a PerCP/Cy5.5, TIGIT PE, CD4 FITC, CD3 APC-Cy7,KLRG1 ALEXA 647, and CD154 BV421 for T cell activation panel 2.Phenotype analysis was done by gating 10,000 to 100,000 cells accordingto FSC/SSC using the Canto II flow cytometer. Data was analyzed usingCytobank software (Cytobank, Inc., Santa Clara, Calif., USA) to createsunburst diagrams and SPADE (Spanning-tree Progression Analysis ofDensity-normalized Events) plots. Gates were set based on fluorescenceminus one (FMO) controls. SPADE plots were generated with the group ofcells, characterized in a form of related nodes based on the expressionlevel of surface markers. CD4⁺ and CD8⁺ TIL subsets were determinedbased on CD3⁺ gating, and trees were generated. Sunburst visualizationsare shown in FIG. 44 and FIG. 45. FIG. 44 shows that TILs expandedagainst aEM3 aAPCs maintained the CD8⁺ phenotype when compared to thesame TILs expanded against PBMC feeders. FIG. 45 shows the results of asecond batch of TILs from a different patient expanded against aEM3aAPCs, where a clear increase of CD8⁺ cells (65.6%) is seen incomparison to the results from expansion using PBMC feeders (25%).

The CD4 and CD8 SPADE tree of TILs expanded with aEM3 aAPCs or PBMCfeeders using CD3⁺ cells is shown in FIG. 46 and FIG. 47. The colorgradient is proportional to the mean fluorescence intensity (MFI) ofLAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without beingbound by theory, the results show that TILs expanded with aEM3 aAPCs hadundergone activation, but there was no difference in MFI between theaEM3 aAPCs and PBMC feeders, indicating that the aEM3 aAPCs effectivelyreplicate the phenotypic results obtained with PBMC feeders.

Spare respiratory capacity (SRC) and glycolytic reserve were alsoevaluated for TILs expanded with aEM3 aAPCs in comparison to PBMCfeeders, with results shown in FIG. 48 and FIG. 49. The Seahorse XF CellMito Stress Test measures mitochondrial function by directly measuringthe oxygen consumption rate (OCR) of cells, using modulators ofrespiration that target components of the electron transport chain inthe mitochondria. The test compounds (oligomycin, FCCP, and a mix ofrotenone and antimycin A, described below) are serially injected tomeasure ATP production, maximal respiration, and non-mitochondrialrespiration, respectively. Proton leak and spare respiratory capacityare then calculated using these parameters and basal respiration. Eachmodulator targets a specific component of the electron transport chain.Oligomycin inhibits ATP synthase (complex V) and the decrease in OCRfollowing injection of oligomycin correlates to the mitochondrialrespiration associated with cellular ATP production. Carbonyl cyanide-4(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent thatcollapses the proton gradient and disrupts the mitochondrial membranepotential. As a result, electron flow through the electron transportchain is uninhibited and oxygen is maximally consumed by complex IV. TheFCCP-stimulated OCR can then be used to calculate spare respiratorycapacity, defined as the difference between maximal respiration andbasal respiration. Spare respiratory capacity (SRC) is a measure of theability of the cell to respond to increased energy demand. The thirdinjection is a mix of rotenone, a complex I inhibitor, and antimycin A,a complex III inhibitor. This combination shuts down mitochondrialrespiration and enables the calculation of nonmitochondrial respirationdriven by processes outside the mitochondria.

FIG. 50 illustrates a mitochondrial stain of Live TILs expanded againstPBMC feeders or aEM3 aAPCs. MitoTracker dye stains mitochondria in livecells and its accumulation is dependent upon membrane potential. TILsexpanded against PBMC feeders or aEM3 were stained L/D Aqua followed byMitoTracker red dye. The data show MitoTracker positive (MFI) cellsgated on live population,

Example 7—Comparison of Engineered MOLM-14 (aMOLM14) and EM-3 (aEM3)aAPCs

TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs, as describedin the previous examples, were assessed for functional activity usingthe BRLA for cytotoxic potency. The P815 BRLA is described in detail inExample 9. The results are shown in FIG. 51 and FIG. 52, and show thatTILs expanded with aAPCs have similar functional properties (andexpected clinical efficacy) to those expanded with PBMC feeders.

IFN-γ release and Granzyme B release from TILs expanded with PBMCfeeders and aMOLM14 and aEM3 aAPCs as described above was also assessedfollowing overnight stimulation with microbeads coated withanti-CD3/CD28/4-1BB. The IFN-γ release results are shown in FIG. 53 andFIG. 54, and the Granzyme B release results are shown in FIG. 55 andFIG. 56. Significant and surprising increases in IFN-γ release andGranzyme B release were observed for TILs expanded with aEM3 aAPCsrelative to those expanded with PBMC feeders, but not for TILs expandedby aMOLM14 aAPCs. Without being bound by theory, this suggests that TILscultured with aEM3 aAPCs may be more active in vivo as a cancer therapy.Most other differences observed were not statistically significant.

The results of TIL expansions with aEM3 and aMOLM14 aAPCs are summarizedin Table 9.

TABLE 9 Summary of TIL expansion results with aAPCs. Fold ExpansionRelative CD8 (%) CD4 (%) Relative Relative aAPC TIL# PBMC aAPC expansionPBMC aAPC PBMC aAPC CD8 CD4 aMOLM14 M1032-T2 2112 1936 0.92 53 65 44 271.226 0.614 M1033-T6 1761 1598 0.91 50 57 36 40 1.140 1.111 M1021T-52053 2024 0.99 91 82 8 17 0.901 2.125 M1030T-4  860  853 0.99 46 78 5112 1.696 0.235 M1045  858*  758* 0.88 — — — — — — M1021T-1 1866 16200.87 — — — — — — M1032T-1 2423 2049 0.85 — — — — — — M1042 1278 17041.33  8  8 88 89 0.919 1.015 M1043 1601 1587 0.99 90 87 5  5 0.968 0.947aEM3 M1054 2058 1647 0.80 98 96 2  2 0.981 1.400 M1055  729 1533 2.10 2566 70 31 2.694 0.441 M1021T-1 2985 2805 0.94 87 75 10 20 0.862 2.000M1045 1336 1047 0.78 — — — — — —

Example 8—Preparation of Master Cell Banks for aEM3 and aMOLM14 aAPCs

aEM3 and aMOLM14 aAPCs may be grown in the following media compositionsto produce master cell banks, which may be further grown in this mediafor supply of aAPCs: 500 mL of Dulbecco's Modified Eagle Medium DMEM/F12(Sigma-Aldrich, St. Louis, Mo., USA), 50 mL fetal bovine serum (FBS)Heat Inactivated (HI) (Hyclone); 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) (LifeTechnologies); 1× Primocin (Invivogen); 1× Plasmocin (Invivogen), and1×2-mercaptoethanol (Life Technologies).

The aAPCs described herein, including aEM3 and aMOLM14 aAPCs, may alsobe grown from a master cell bank using any suitable method known in theart for the growth of cells. In an embodiment, aAPCs are thawed and arethen expanded in a medium of 80-90% RPMI 1640+10-20% h.i. FBS (fetalbovine serum) by splitting saturated culture 1:2 to 1:3 every 2-3 days,seeding out at about 0.5-1×10⁶ cells/mL in 24-well plates, andmaintaining at about 0.5-1.5×10⁶ cells/mL, with incubation at 37° C. and5% CO₂.

Further steps that may be employed to use the aAPCs of certainembodiments of the present invention in the production of humantherapies are known in the art and include cell line characterization(HLA high resolution typing); cytokine release testing; testing of humanserum to replace FBS to grow aAPC; testing freezing media to freezeaAPCs; master cell banking (including raw material testing and stabilitytesting); standardization of irradiation (including irradiation dose(1000, 3000, 5000, 10000, 15000 rad), fresh versus frozen aAPCs, andwith/without TILs); stability of aAPC; development of a panel toevaluate the contamination of aAPCs; development of molecular biologyassays (qPCR, DNA sequencing); testing of TIL expansions from differenttumor types, including melanoma, cervical, and head and neck cancer(using a G-Rex 5M); potency, purity, and identity testing; mycoplasmaand sterility assays; microbiological testing (USP/EP sterility,bioburden and endotoxin assays); and adventitious viral agent testing.

Example 9—Methods of Expanding TILs and Treating Cancer with ExpandedTILs

TILs may be expanded using the aAPCs of certain embodiments of thepresent invention, such as aEM3 and aMOLM14 aAPCs, using any of theexpansion methods described herein. For example, a method for expandingTILs is depicted in FIG. 57. The expansion of TILs using aAPCs may befurther combined with any method of treating cancer in a patientdescribed herein. A method for expanding TILs and treating a patientwith expanded TILs, wherein the expansion makes use of aAPCs (includingaEM3 and aMOLM14 aAPCs), is shown in FIG. 58.

Example 10—P815 Bioluminescent Redirected Lysis Assay

In this example, the development of a surrogate target cell line toevaluate the lytic potential of TILs in a Bioluminescent RedirectedLysis Assay (BRLA) is described. The BRLA enables assessment of T cellmediated killing in the absence of autologous tumor cells. Cytolyticactivity can be assessed with and without engaging the T cell receptorin one to four hours, assessing T cell killing engaging the T cellreceptor and without so-called lymphokine activated killer activities(LAK).

Mouse mastocytoma P815 cells expressing the endogenous CD16 Fc receptorcan bind anti-CD3c (OKT-3), providing a potent TCR activation signal asa target cell line. The P815 Clone G6 was transduced with a lentiviralvector based on eGFP and firefly luciferase, sorted and cloned using theBD FACSAria II. Clone G6 was selected based on eGFP intensity analyzedusing an Intellicyt iQue Screener. Target cells and TILs of interestwere co-cultured+/−OKT-3 to assess TCR activation (specific killing) ornon-specific (lymphokine activated killing, LAK) respectively. Following4 hours of incubation, firefly luciferin((4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylicacid, commercially available from multiple sources) was added to thewells and incubated for 5 minutes. Bioluminescence intensity was readusing a luminometer. Percent cytotoxicity and survival were calculatedusing the following formula: % Survival=(experimentalsurvival−minimum)/(maximum signal−minimum signal)×100; %Cytotoxicity=100−(% Survival). Interferon gamma release in the mediasupernatant of co-cultured TILs was analyzed by ELISA, and LAMP1(CD107a, clone eBioH4A3) expression on TILs was analyzed on a flowcytometer to evaluate the cytotoxic potency of TILs.

Results are shown in FIG. 59 to FIG. 75. FIG. 59 illustrates percenttoxicity of TIL batch M1033T-1 co-cultured with P815 Clone G6 (with andwithout anti-CD3) at individual effector:target ratios by BRLA. FIG. 60illustrates enzyme-linked immunosorbent assay (ELISA) data showing theamount of IFN-γ released against different ratios of effector to targetcells. FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 whenco-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of1:1 effector to target cells for 4 hours and 24 hours co-culture.

The results were confirmed using a second TIL batch as shown in FIG. 62,which illustrates BRLA for TIL batch M1030. The cytotoxicity (measuredas LU₅₀/1×10⁶ TIL) by BRLA is 26±16. FIG. 63 illustrates the results ofa standard chromium release assay for TIL batch M1030. The cytotoxicity(measured as LU₅₀/1×10⁶ TIL) by chromium release assay is 22.

Results were further confirmed using a third TIL batch. FIG. 64illustrates BRLA results for TIL batch M1053, showing lytic units of theTILs by BRLA as 70±17. FIG. 65 illustrates the results of a standardchromium release assay for TIL batch M1053, showing lytic unit of theTILs by chromium assay as 14±5. Comparison of two assay results showsthe comparable performance of the BRLA result to the chromium releaseassay result.

FIG. 66 illustrates the linear relationship between IFN-γ release andcytotoxic potential of TILs. FIG. 67 illustrates ELISpot results forIFN-γ. FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053.FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030. FIG. 70illustrates ELISpot data showing Granzyme B release by M1053T andM1030T. FIG. 71 illustrates enzymatic Granzyme B release for TIL batchM1053. FIG. 72 illustrates enzymatic Granzyme B release for TIL batchM1030. FIG. 73 illustrates ELISpot data showing TNF-α release by M1053Tand M1030T. FIG. 74 illustrates enzymatic TNF-α release for TIL batchM1053. FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030.The data in FIG. 66 to FIG. 76 confirms the potency of these batches ofTILs as also shown by the BRLA.

In conclusion, the BRLA requires no radionuclides and is as efficientand sensitive as traditional cytotoxicity assays. Flow cytometricassessment of Lampl expression on TILs at individual time pointsdemonstrates degranulation of cytotoxic T cells relative to the potencyshown by BRLA. The BRLA demonstrates similar to better potency thanstandard chromium release assay. BRLA also enables evaluation of thepotency of TIL lytic activity. Comparison of BRLA with chromium releaseassay shows the efficiency and reliability of BRLA. BRLA has a linearrelationship with IFNγ release by TILs. Release assay of IFN-γ, TNFα andGranzyme B by ELISpot is consistent with the cytotoxic efficiency of theTILs evaluated by BRLA.

Example 11—Process for Weaning EM3 Cells from FBS to hAB Serum

In order to avoid reactivity, some cell lines may need to be weaned fromone medium to another. Here, EM3 cells are weaned from FBS to hAB serumto avoid reactivity. As shown in FIG. 76, aEM3 cells were successfullyweaned off of FBS to hAB serum.

Example 12—Freezing Media Formulation Optimization

To cryobank EM3 cells cultured as described herein, methods werefreezing media formulation were optimized. As shown in FIG. 77, threefreezing media were used and their effect on cell numbers were counted.The cell media utilized included CryStor 10 (Biolife Solutions (CS10))(A), hAB [90%] and DMSO [10%] (B), and hAB [20%] with DMSO [10%] andcDMEM2 [70%] (C). FIG. 77 demonstrates that the formulation of human ABserum (90%) and DMSO (10%) provided for unexpectedly increased EM3 cellnumbers after 3 days of recovery.

Example 13—Growth of aEM3 Cells in GREX Flasks

aEM3 cells were cultured in gas permeable cell culture flasks (i.e.,GREX flasks (Wilson Wolf Manufacturing)) and the effect on cell doublingtime was observed over an 8 day time course. As shown in FIG. 78, theGREX flasks provided for rapid growth of aEM3 cells.

Example 14—Flow Panel Analysis to Determine aEM3 Cell Purity

To determine the purity of cells cultured according to the processesdescribed herein, a flow panel analysis was used to determine the purityof aEM3 aAPCs. The results of such analysis are described in FIGS. 79and 80. As shown in FIG. 80, before sorting aEM3 cell populations were53.5% and 43.2% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively.Postsorting, cell populations was improved to 96.8% and 96.3% eGFP+ foraEM3 7C12 and aEM3 8B5 cells, respectively (FIG. 80).

Example 15—aEM3 Feeder Cells as an Alternative to PBMC Feeders

As described herein, aEM3 cells may be used as an alternative for PBMCfeeders, resulting in unexpectedly different properties for both TILexpansion process and the resulting TILs. To compare differences incytokine expression, PBMCs and aEM3 cells were stimulated by treatmentwith OKT-3. As shown in FIG. 81, aEM3 cells displayed a comparativelydifferent cytokine expression profile as compared to PBMCs.Surprisingly, the aEM3 cells of the present invention provideefficacious TILs (as shown herein) without reproducing the same cytokinesecretion properties of TILs expanded with conventional PBMCs.

Example 16—Comparison Between Complete Media and Serum Free Media TILExpansion

In order to optimize the TIL expansion protocols, several TIL expansionexpirements were peformed as described herein, but with serum free mediarather than complete media (CM1).

In one experiment, tissue fragments were cultured in a single well withCM1 or various serum free media with 300 IU/mL of IL-2. Cells were thencounted on Day 11 before initiating REP. The various serum free mediaused included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), andXvivo-20 (Lonza). As shown in FIG. 82, TIL expansion (PreREP) with CTSprovided increased cell numbers as compared to CM1.

Additionally, tissue fragments were cultured with CM1 or various serumfree media with 6000 IU/mL of IL-2 until Day 11. REP was then initiatedon Day 11 using PBMC feeders, OKT-3, and IL-2, and culture was split onDay 16. Cultures were then terminated at the end of Day 22. The variousserum free media used included Prime CDM (Irvine), CTS Optimizer(ThermoFisher), and Xvivo-20 (Lonza). As shown in FIG. 83 and FIG. 84,when counting cells at Days 11 and Day 22, respectively, TIL expansion(PreREP) with Prime CDM provided increased cell numbers as compared toCM1.

Example 17—Growth of aAPCs in Serum Free Media as Compared toSerum-Based Media

In order to optimize aAPC growth and maintenance protocols in theabsence of serum, aEM3 cells were cultured using various serum freemedia.

aEM3 cells were cultured in 24 well plates at 1×10⁶ cells per well for 3days using general cell culture protocols as described herein, with theexception that that one group of cells were provided with serum-basedmedia (cDMEM (10% hSerum) and the other groups of cells were providedwith serum free media. The serum free media utilized for the studyincluded CTS OpTmizer (ThermoFisher), Xvivo 20 (Lonza), Prime-TCDM(Irvine), and XFSM (MesenCult) media. Cells were then counted on Day 3.

As shown in FIG. 85, CTS OpTmizer and Prime-TCDM serum free mediaprovided cell growth that was comparable to serum-based media (i.e.,cDMEM (10% hSerum). Therefore, serum free media is an effectivealternative for growing and maintaining aAPCs as comapred to serum-basedmedia.

Example 18—Propagation, Maintenance, and Cryopreservation of aAPCs

In this example, procedures are provided for the preparation andpreservation of aAPCs. Specifically, aEM3 cells from a cell linedesignated TIL-Rs3 were propagated and cryopreserved.

Thawing and recovery of aEM3 cells may be accomplished using thefollowing non-limiting procedure. Cyropreserved aEM3 cells are warmedslowly in pre-warmed media (37° C.) that is prepared from CTS OpTmizerBasal Media (Thermo Fisher), CTS OpTmizer Cell Supplement (ThermoFisher), Gentamicin (Lonza), and Glutamax (Life Technologies). Thesuspended cells are then centrifuged at 1500 rpm for 5 minutes at 4° C.The resulting supernatant is discarded and the remaining aEM3 cells areresuspended in the foregoing media and plated (5×10⁶ cells/10 mL perwell of a 6 well plate).

Propagation of aEM3 cells may be accomplished using the followingnon-limiting procedure. Aliquots of the foregoing media are prepared ingas permeable cell culture flasks (i.e., GREX 10 flasks (Wilson WolfManufacturing)). The plated aEM3 cells are washed by centrifugation(i.e., 1500 rpm for 5 minutes at 4° C.), resuspended in media, and addedto the GREX flasks at cell density of 1-2×10⁶ cells/mL. The aEM3 cellsuspension was diluted with 30 mL of media and the GREX flasks were thenincubated for 3-4 days at 37° C. under CO₂. After 3-4 days, the GREXflasks were removed from the incubator and placed in a biological safetycabinet (BSC). The cultured aEM3 cells are carefully extracted from theGREX flasks by pipette and the resulting extraction is centrifuged toprovide the increased number of aEM3 cells, which may be resuspended ata cell density of 10-20×10⁶ cells per GREX 10 flask.

An alternative cryopreservation of aEM3 cells may be accomplished usingthe following non-limiting procedure. The foregoing GREX 10 flaskscontaining the aEM3 cells are removed from the incubator and placed in aBSC. The cultured aEM3 cells are carefully extracted from the GREXflasks by pipette and the resulting extraction is centrifuged to providethe increased number of aEM3 cells, which is resuspended in a volume ofCryStor 10 (Biolife Solutions) to provide a concentration of 10-100×10⁶cells/vial in cryovials. The aEM3 cell suspensions may be placed in afreezing container and transferred to a −80° C. freezer.

Example 19—Demonstration of Rapid Recovery of aEM3 Cells FollowingCryopreservation

aEM3 cells from the TIL-R3 cell line (1-2×10⁶ cells) were cryopreservedaccording to the procedure set forth in Example 18 using CS-10cryopreservation media. Vials of such cells were then thawed and thecells were counted. Cell counts were taken pre-freeze, post-thaw, and 3days after thaw (i.e., Post-Thaw Recovery). As shown in FIG. 86 and FIG.87, the total live cell counts recovered rapidly post thaw in twoseparate experiments.

TIL-R3 cells (1×10⁶ cells) were thawed (Day 3 post-thaw) and plated at adensity of 0.5×10⁶/cm² in each well of a 24 well plate. On day 0 and 3,viable cells were counted and recorded. On the first passage (Day 6),cells were split at the density of 2×10⁶ cells/cm² or 0.5×10⁶ cells/cm².At the end of the first passage, a cell count was performed. Theresulting cell counts are shown in FIG. 88, which demonstrate both arecovery phase post-thaw and a growth phase.

Furthermore, TIL-R3 cells (20×10⁶ cells) were cultured at a density of2×10⁶/cm² in GREX 10 flasks according to the procedure described inExample 18. On days 4 and 8, live cells were counted and recorded. Theresulting cell counts are shown in FIG. 89, which demonstrates a growthphase for the cells following cryopreservation that reaches a plateaubetween days 4 and 8 when the cells reached a density of 13.9×10⁶cells/cm′.

Example 20—CD8 Skewness, Expansion Performance, and CD3 Contamination ofTILs Cultured with aEM3 aAPCs

Fifteen different PreREP TIL lines (0.4×10⁵ cells) were co-cultured witheither aEM3 aAPCs (as described herein) or PBMC feeders (10×10⁶), OKT3(30 ng/mL) and IL-2 (3000 IU/mL) and cultures were split on Day 5 using6 well Grex plates. Cultures were sampled at day 11 and analyzed by flowcytometry. The relative ratio of CD8⁺ cells was calculated by theformula (CD8% aEM3)/(CD8% PBMC). The results shown in FIG. 91 indicatethat TILs cultured with aEM3 cells surprisingly promote CD8⁺ skewing andand an improved TIL product. Additional results of these experiments areshown in FIG. 92, FIG. 93, and FIG. 94, where the results shown thatTILs cultured with aEM3 aAPCs displayed comparable expansion and lessnon-CD3+ cell contamination in comparison to TILs cultured with PBMCfeeders.

Example 21—Telomere Length Measurement

Genomic DNA was isolated from pre-REP or post-REP (magnetic bead sortedfor CD3⁺) TILs for a qPCR (quantitative polymerase chain reaction) assayto measure telomere length. The real time qPCR method is described inCawthon, Nucleic Acids Res. 2002, 30(10), e47; and Yang, et al.,Leukemia, 2013, 27, 897-906. Briefly, the telomere repeat copy number tosingle gene copy number (T/S) ratio was determined using an PCR thermalcycler (Bio-Rad Laboratories, Inc.) in a 96-well format. Ten ng ofgenomic DNA was used for either the telomere or hemoglobin (hgb) PCRreaction and the primers used were as follows:

Tel-1b primer (SEQ ID NO: 40)(CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT); Tel-2b primer(SEQ ID NO: 41) (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT);hgb1 primer (SEQ ID NO: 42) (GCT TCT GAC ACA ACT GTG TTC ACT AGC); andhgb2 primer (SEQ ID NO: 43) (CAC CAA CTT CAT CCA CGT TCA CC).

All samples were analyzed by both the telomere and hemoglobin reactions,and the analysis was performed in triplicate on the same plate. Inaddition to the test samples, each 96-well plate contained a five-pointstandard curve from 0.08 ng to 250 ng using genomic DNA isolated fromthe 1301 human T-cell leukemia cell line (available from Sigma andATCC). The T/S ratio (−dCt) for each sample was calculated bysubtracting the median hemoglobin threshold cycle (Ct) value from themedian telomere Ct value. The relative T/S ratio (−ddCt) was determinedby subtracting the T/S ratio of the 10.0 ng standard curve point fromthe T/S ratio of each unknown sample.

Results are shown in FIG. 95. Each data point shown is the medianmeasurement of relative T/S ratio. The results indicate that TILscultured with aEM3 maintain their telomere length.

1.-99. (canceled)
 100. A method of treating a subject having cancer witha population of lymphocytes, the method comprising: (a) obtaining afirst population of lymphocytes from a tumor resected from a patient;(b) performing an initial expansion of the first population oflymphocytes in a first cell culture medium to obtain a second populationof lymphocytes, wherein the second population of lymphocytes is at least5-fold greater in number than the first population of lymphocytes, andwherein the first cell culture medium comprises IL-2; (c) performing arapid expansion of the second population of lymphocytes in a second cellculture medium to obtain a third population of lymphocytes, wherein thethird population of lymphocytes is at least 50-fold greater in numberthan the second population of lymphocytes after about 7 days from thestart of the rapid expansion; and wherein the second cell culture mediumcomprises IL-2 or OKT-3, (d) transducing one of the first, the second,or the third population of lymphocytes with one or more viral vectorscomprising a nucleic acid encoding a cell surface binding molecule,and/or one or more nucleic acids encoding one or more costimulatorymolecules, wherein the first, the second, or the third population oflymphocytes expresses the cell surface binding molecule and the one ormore costimulatory molecules; and (e) administering a therapeuticallyeffective portion of the third population of lymphocytes to a subjectwith the cancer.
 101. The method of claim 100, wherein the lymphocytescomprise tumor-infiltrating lymphocytes (TILs).
 102. The method of claim100, wherein the one or more costimulatory molecules are independentlyselected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD1a,CD1b, CD1c, CD1d, CD2, CD3γ, CD3δ, CD3∈, CD4, CD5, CD6, CD7, CD8α, CD8β,CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a,CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD3δ, CD37, CD38, CD39, CD40,CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47,CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53,CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P,CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68,CD69, CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79α, CD79β,CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91,CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102,CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD114,CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b,CD122, CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b, CDw130,CDw131, CD132, CD133, CD135, CD136, CD138, CD139, CD140a, CD140b, CD141,CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151,CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162,CD163, CD164, CD165, CD166, and TCRξ.
 103. The method of claim 102,wherein the one or more costimulatory molecules are independentlyselected from the group consisting of CD28, 4-1BB (CD137), and OX40(CD134).
 104. The method of claim 100, wherein the cell culture mediumcomprises IL-2.
 105. The method of claim 104, wherein the IL-2 is at aninitial concentration of about 3000 IU/mL.
 106. The method of claim 100,wherein the cell culture medium comprises OKT-3 antibody.
 107. Themethod of claim 106, wherein the OKT-3 antibody is at an initialconcentration of about 30 ng/mL.
 108. The method of claim 100, whereinthe rapid expansion is performed over a period not greater than 14 days.109. The method of claim 100, wherein one or both of the initialexpansion and the rapid expansion is performed using a gas permeablecontainer.
 110. The method of claim 100, wherein the cancer is selectedfrom the group consisting of melanoma, ovarian cancer, cervical cancer,non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breastcancer, cancer caused by human papilloma virus, head and neck cancer,renal cancer, renal cell carcinoma, pancreatic cancer, and glioblastoma.111. The method of claim 100, wherein the one or more viral vectorscomprise a lentiviral vector.
 112. The method of claim 100, wherein therapid expansion is performed using a population of antigen presentingcells (APCs).
 113. The method of claim 112, wherein the population ofAPCs expands the population of lymphocytes by at least 50-fold over aperiod of about 7 days.
 114. The method of claim 112, wherein thepopulation of APCs endogenously express HLA-AB/C, ICOS-L, and CD58. 115.The method of claim 112, wherein the population of APCs are transducedto express an anti-OKT-3 antibody scFv binding domain.
 116. The methodof claim 112, wherein the ratio of the second population of lymphocytesto the population of APCs is between about 1 to 200 and about 1 to 400.117. The method of claim 100, wherein the population of lymphocytes arecryopreserved.
 118. The method of claim 101, wherein the TILs arecryopreserved.
 119. The method of claim 100, wherein the cell surfacebinding molecule comprises a single chain fragment variable (scFv)binding domain.